Protein Processing in Plant Mitochondria Compared to Yeast and Mammals

Abstract

Limited proteolysis, called protein processing, is an essential post-translational mechanism that controls protein localization, activity, and in consequence, function. This process is prevalent for mitochondrial proteins, mainly synthesized as precursor proteins with N-terminal sequences (presequences) that act as targeting signals and are removed upon import into the organelle. Mitochondria have a distinct and highly conserved proteolytic system that includes proteases with sole function in presequence processing and proteases, which show diverse mitochondrial functions with limited proteolysis as an additional one. In virtually all mitochondria, the primary processing of N-terminal signals is catalyzed by the well-characterized mitochondrial processing peptidase (MPP). Subsequently, a second proteolytic cleavage occurs, leading to more stabilized residues at the newly formed N-terminus. Lately, mitochondrial proteases, intermediate cleavage peptidase 55 (ICP55) and octapeptidyl protease 1 (OCT1), involved in proteolytic cleavage after MPP and their substrates have been described in the plant, yeast, and mammalian mitochondria. Mitochondrial proteins can also be processed by removing a peptide from their N- or C-terminus as a maturation step during insertion into the membrane or as a regulatory mechanism in maintaining their function. This type of limited proteolysis is characteristic for processing proteases, such as IMP and rhomboid proteases, or the general mitochondrial quality control proteases ATP23, m-AAA, i-AAA, and OMA1. Identification of processing protease substrates and defining their consensus cleavage motifs is now possible with the help of large-scale quantitative mass spectrometry-based N-terminomics, such as combined fractional diagonal chromatography (COFRADIC), charge-based fractional diagonal chromatography (ChaFRADIC), or terminal amine isotopic labeling of substrates (TAILS). This review summarizes the current knowledge on the characterization of mitochondrial processing peptidases and selected N-terminomics techniques used to uncover protease substrates in the plant, yeast, and mammalian mitochondria.

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fpls-13-824080 January 27, 2022 Time: 15:45 # 1
REVIEW
published: 02 February 2022
doi: 10.3389/fpls.2022.824080
Edited by:
Emmanuelle Graciet,
Maynooth University, Ireland
Reviewed by:
Pitter F. Huesgen,
Julich Research Center, Helmholtz
Association of German Research
Centres (HZ), Germany
Shaobai Huang,
The University of Western Australia,
Australia
*Correspondence:
Malgorzata Heidorn-Czarna
malgorzata.czarna@uwr.edu.pl
Specialty section:
This article was submitted to
Plant Proteomics and Protein
Structural Biology,
a section of the journal
Frontiers in Plant Science
Received: 28 November 2021
Accepted: 12 January 2022
Published: 02 February 2022
Citation:
Heidorn-Czarna M, Maziak A and
Janska H (2022) Protein Processing
in Plant Mitochondria Compared
to Yeast and Mammals.
Front. Plant Sci. 13:824080.
doi: 10.3389/fpls.2022.824080
Protein Processing in Plant
Mitochondria Compared to Yeast and
Mammals
Malgorzata Heidorn-Czarna*, Agata Maziak and Hanna Janska
Department of Cellular Molecular Biology, Faculty of Biotechnology, University of Wrocław, Wrocław, Poland
Limited proteolysis, called protein processing, is an essential post-translational
mechanism that controls protein localization, activity, and in consequence, function.
This process is prevalent for mitochondrial proteins, mainly synthesized as precursor
proteins with N-terminal sequences (presequences) that act as targeting signals and
are removed upon import into the organelle. Mitochondria have a distinct and highly
conserved proteolytic system that includes proteases with sole function in presequence
processing and proteases, which show diverse mitochondrial functions with limited
proteolysis as an additional one. In virtually all mitochondria, the primary processing
of N-terminal signals is catalyzed by the well-characterized mitochondrial processing
peptidase (MPP). Subsequently, a second proteolytic cleavage occurs, leading to more
stabilized residues at the newly formed N-terminus. Lately, mitochondrial proteases,
intermediate cleavage peptidase 55 (ICP55) and octapeptidyl protease 1 (OCT1),
involved in proteolytic cleavage after MPP and their substrates have been described
in the plant, yeast, and mammalian mitochondria. Mitochondrial proteins can also be
processed by removing a peptide from their N- or C-terminus as a maturation step
during insertion into the membrane or as a regulatory mechanism in maintaining their
function. This type of limited proteolysis is characteristic for processing proteases,
such as IMP and rhomboid proteases, or the general mitochondrial quality control
proteases ATP23, m-AAA, i-AAA, and OMA1. Identification of processing protease
substrates and defining their consensus cleavage motifs is now possible with the help
of large-scale quantitative mass spectrometry-based N-terminomics, such as combined
fractional diagonal chromatography (COFRADIC), charge-based fractional diagonal
chromatography (ChaFRADIC), or terminal amine isotopic labeling of substrates (TAILS).
This review summarizes the current knowledge on the characterization of mitochondrial
processing peptidases and selected N-terminomics techniques used to uncover
protease substrates in the plant, yeast, and mammalian mitochondria.
Keywords: protein processing, proteases, mitochondria, N-terminomics, COFRADIC, ChaFRADIC, TAILS , limited
proteolysis
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Heidorn-Czarna et al. Mitochondrial Protein Processing
INTRODUCTION
Mitochondria evolved approximately 1.5 billion years ago as
double-membrane organelles originating from the bacterial
phylum α-Proteobacteria entering a stable endosymbiosis with
the host cell (Gray et al., 1999;Gray, 2012). Over the years
of evolution, the mitochondrial genome (mitochondrial DNA,
mtDNA) has been drastically reduced by relocating most of
the genetic information in the nucleus. The mitochondrial
genome of plant Arabidopsis thaliana encodes 33 proteins
(Unseld et al., 1997;Sloan et al., 2018). In comparison,
there are only 13 proteins in humans (Anderson et al., 1981;
Capt et al., 2016) and eight proteins in yeast Saccharomyces
cerevisiae encoded by the mtDNA (Foury et al., 1998;Malina
et al., 2018). In consequence, most mitochondrial proteins
are synthesized on cytosolic ribosomes and imported post-
translationally to the mitochondria. Delivery of mitochondrial
precursor proteins to the organelle and sorting them into one
of the four mitochondrial subcompartments (outer membrane,
OM; intermembrane space, IMS; inner membrane, IM; matrix)
is achieved by mitochondrial targeting signals such as cleavable
N-terminal presequences, several non-cleavable internal and
C-terminal signal sequences or cysteine-rich motifs (Table 1)
(Schleiff and Becker, 2011). These targeting signals are recognized
by specific receptors of the protein import machinery localized
in the outer and inner membranes allowing for the correct
protein targeting and sorting in the mitochondria (Ghifari
et al., 2018;Pfanner et al., 2019). Most mitochondrial proteins
contain N-terminal targeting sequences, which are cleaved
upon import into mitochondria, generating functional proteins
(Teixeira and Glaser, 2013;Poveda-Huertes et al., 2017;
Ghifari et al., 2019).
In contrast to complete protein degradation, protein
processing, often referred to as limited proteolysis, is an
essential biological mechanism controlling protein activity,
location, or function. Removing the N-terminal mitochondrial
presequences is the most common way of protein processing
in the mitochondria. However, the processing of mitochondrial
proteins can also be achieved by cleaving a peptide from the
N- or C-terminus of a protein as a precise maturation step
during insertion of a nuclear or mitochondrially encoded
protein into the membrane or as a regulatory mechanism in
TABLE 1 | Targeting sequences of nuclear-encoded mitochondrial proteins.
Destination Signal
OM •Non-cleavable N-terminal, internal, C-terminal, or multiple anchors.
•Non-cleavable β-signal.
IMS •Cleavable N-terminal presequence with cleavable hydrophobic
sorting signal.
IM •Cleavable N-terminal presequence with non-cleavable hydrophobic
sorting signal.
•Non-cleavable multiple hydrophobic signals.
•Non-cleavable internal presequence-like signal.
•Cysteine-rich motif.
Matrix •Cleavable N-terminal presequence.
OM, outer membrane; IMS, intermembrane space; IM, inner membrane. Based
on Schleiff and Becker (2011).
maintaining mitochondrial function. Recently, the identification
in the Arabidopsis mitochondrial ribosome fractions of several
proteases involved in protein maturation and degradation has
suggested that processing of nascent polypeptides may also occur
at mitochondrial ribosomes (Rugen et al., 2019).
Mitochondria possess a distinct and highly conserved
proteolytic system that includes proteases with sole function
in presequence processing and proteases, which show diverse
mitochondrial functions with limited proteolysis as an additional
one. Mitochondrial proteases often do not have one role, and
many show overlapping substrate specificity (Glynn, 2017).
Generally, mitochondrial proteases can be classified based on the
utilization of ATP for their activity, which gives two groups of
proteases: ATP-dependent and ATP-independent (Janska et al.,
2010). Members of processing peptidases are present in both
groups (Table 2). In this review, we will focus predominantly
on processing peptidases and their substrates in the plant, yeast,
and mammalian mitochondria. Additionally, we will highlight
mass spectrometry-based N-terminomics techniques elucidating
mitochondrial protease cleavage sites and uncovering their
proteolytic substrates.
N-TERMINAL SIGNALS TARGETING
PROTEINS INTO MITOCHONDRIA AND
CHLOROPLASTS
In plant and algal cells, the evolution of protein targeting
mechanisms has been especially challenging due to another
type of endosymbiotic organelle, a chloroplast. Most chloroplast
proteins, similarly to mitochondria, are nuclear-encoded,
synthesized as precursors with N-terminal targeting peptides,
and imported to the organelle via the chloroplast-specific
protein import machinery (Shi and Theg, 2013;Wollman, 2016).
Mitochondrial targeting peptides (called presequences) and
the chloroplast ones (called transit peptides) show differences
in total length and primary structure (Bhushan et al., 2006;
Holbrook et al., 2016). Transit peptides are usually longer, with
an average of 56 residues in Arabidopsis (Carrie et al., 2015),
while mitochondrial presequences have an average length of 43
amino acids in Arabidopsis and 45 in Oryza sativa (Huang et al.,
2009;Carrie et al., 2015). In contrast, yeast and mammalian
presequences are shorter and have a length of 30–37 amino acids
(Schneider et al., 1998;Carrie et al., 2015).
Mitochondrial presequences tend to form an amphiphilic
α-helix (von Heijne, 1986;Roise et al., 1988), whereas
chloroplast transit peptides are somewhat unstructured (Bruce,
2001). However, both presequences and transit peptides show
remarkably high similarity at the level of amino acid composition
(Bhushan et al., 2006). Interestingly, it has been demonstrated
that in cells lacking plastids, specific chloroplast transit
peptides deliver proteins into mitochondria (Hurt et al.,
1986a,b;Cleary et al., 2002). There is also an increasing
number of N-terminal dual targeting peptides, called ambiguous
presequences, responsible for distributing dual-targeted proteins
between chloroplasts and mitochondria (Pujol et al., 2007;Carrie
and Whelan, 2013;Baudisch et al., 2014). N-terminal sequence
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Heidorn-Czarna et al. Mitochondrial Protein Processing
TABLE 2 | Mitochondrial proteases involved in limited proteolysis and their substrates in yeast, mammalian, and plant mitochondria.
Protease
(Class)
Yeast Human Plants
Subunits Localization Substrates Subunits Localization Substrates Subunits Localization Substrates
MPP*
(Metalloprotease)
Mas1
Mas2
Matrix Majority of presequence-containing
proteins
(Burkhart et al., 2015)
PMPCB
PMPCA
Matrix Majority of presequence-containing
proteins
(Calvo et al., 2017)
β-MPP
α-MPP
Inner membrane
(integration into the
cytochrome b-c1
complex)
Majority of
presequence-
containing
proteins
ICP55*
(metalloprotease)
Icp55 Inner
membrane-
bound from the
matrix site
Listed in Vögtle et al. (2009) and Venne
et al. (2013)
XPNPEP3 Inner
membrane-
bound from the
matrix site
Putative substrates of human and
mouse Icp55 homolog are listed in
Calvo et al. (2017)
ICP55 Soluble
mitochondrial
fraction
Listed in Carrie
et al. (2015) and
Huang et al. (2015)
OCT1*
(metalloprotease)
Oct1 Matrix Listed in Vögtle et al. (2009) and Vögtle
et al. (2011)
MIP Matrix Putative substrates of human and
mouse MIP are listed in Calvo et al.
(2017);
Cox4, Prx V, OXA1L
(Pulman et al., 2021;Sim et al.,
2021)
OCT1 Membrane-bound Listed in Carrie
et al. (2015)
IMP* (serine
protease)
Imp1
Imp2
Som1
Inner
membrane
Cox2, Cyb2, Mcr1, Gut2, Ptc5, Mgr2,
Mcp3
(Nunnari et al., 1993;Bauer et al.,
1994;Hahne et al., 1994;Chen et al.,
1999;Esser et al., 2004;Ieva et al.,
2013;Sinzel et al., 2016)
cyt c1, Prx1
(Jan et al., 2000;Gomes et al., 2017)
IMMP1L
IMMP2L
Inner
membrane
Smac/DIABLO, (Burri et al., 2005).
cyt c1, FAD-dependent
glycerol-3-phosphate
dehydrogenase (Luo et al., 2006)
IMP1a
IMP2
Membrane-bound Unknown
ATP23*
(metalloprotease)
Atp23 Intermembrane
space
Atp6
(Osman et al., 2007;Zeng et al., 2007)
ATP23 Intermembrane
space
Unknown ATP23 Soluble
mitochondrial
fraction
Unknown
Rhomboid
protease* (serine
protease)
Pcp1 (Rbd1) Inner
membrane
Ccp1, Mgm1 (Esser et al., 2002;Herlan
et al., 2003;Schäfer et al., 2010)
PARL Inner
membrane
Pink1, PGAM5, Smac/DIABLO,
TTC19
(Jin et al., 2010;Sekine et al.,
2012;Saita et al., 2017;Spinazzi
et al., 2019)
RBL12 Inner membrane Unknown
m-AAA**
(metalloprotease)
Yta10
Yta12
Inner
membrane
Ccp1, MrpL32, Oxa1, Ilv2
(Esser et al., 2002;Käser et al., 2003;
Nolden et al., 2005;Dasari and Kölling,
2016)
SPG7
AFG3L2
Inner
membrane
MrpL32, OPA1, OMA1
(Nolden et al., 2005;Ishihara et al.,
2006;Almajan et al., 2012;
Consolato et al., 2018)
FTSH3
FTSH10
Inner membrane AtL32
(Kolodziejczak
et al., 2018)
i-AAA**
(metalloprotease)
Yme1 Inner
membrane
Atg32, Ilv2
(Wang et al., 2013;Dasari and Kölling,
2016)
YME1L1 Inner
membrane
OPA1
(Ehses et al., 2009;Head et al.,
2009;Anand et al., 2014)
FTSH4 Inner membrane Unknown
OMA1*
(Metalloprotease)
Oma1 Inner
membrane
Unknown OMA1 Inner
membrane
OPA1, DELE1
(Ehses et al., 2009;Head et al.,
2009;Anand et al., 2014;Xiao
et al., 2014;Guo et al., 2020)
OMA1 Membrane-bound Unknown
MPP, mitochondrial processing peptidase; ICP55, intermediate cleavage peptidase of 55 kDa; OCT1, octapeptidyl aminopeptidase 1; IMP, inner membrane peptidase; ATP23, ATP synthase 23; m-AAA, matrix-
ATPase associated with a variety of cellular activities; i-AAA, intermembrane space-ATPase associated with a variety of cellular activities; OMA1, Overlapping with the m-AAA protease 1. *ATP-independent protease;
**ATP-dependent protease.
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analyses of dual-targeted proteins from Arabidopsis showed their
average length of 59 amino acids, which is significantly longer
than typical mitochondrial presequences (Carrie et al., 2015).
Studies of mitochondrial and chloroplast signal peptides
revealed distinct differences in the first 16 amino acids and that
the N-terminal end of presequences is enriched with arginine
(Arg, R) residues (Bhushan et al., 2006). The N-terminal arginines
have been previously shown to be essential in mitochondrial
protein import in plants (Duby et al., 2001). In a recent study,
Lee et al. (2019) revealed that within the first 12 amino acids
of a presequence, four Arg residues, referred to as the 4-Arg
motif, are critical for delivering a protein to mitochondria
(Figure 1). Removing the 4-Arg motif changed the targeting
specificity from mitochondria to chloroplasts, whereas adding
the 4-Arg motif to the N-terminal region of transit peptides
blocked targeting a protein to chloroplasts. On the contrary,
moderate hydrophobicity at the N-terminal region of transit
peptides was sufficient to specify protein import into chloroplasts.
Furthermore, the study also revealed that the chimeric targeting
peptide containing the N-terminal region of a chloroplast transit
peptide and a complete mitochondrial presequence would specify
targeting a protein to both organelles (Figure 1) (Lee et al., 2019;
Lee and Hwang, 2020). Similar to the earlier findings (Pujol
et al., 2007), Lee et al. (2019) have also shown that the presence
of conserved Arg residues determines mitochondrial targeting
by both mitochondrial and dual-targeting peptides. Further in-
depth surveys are still necessary to understand the evolution of
dual-targeted sequences leading to a protein targeting into both
mitochondria and chloroplasts (Lee and Hwang, 2020).
MAIN MITOCHONDRIAL PROCESSING
PROTEASES
Cleavage of mitochondrial targeting signal is carried out by
different processing proteases localized in the mitochondrial
matrix and at the inner membrane. The N-terminal presequences
of mitochondrial proteins are often removed in the matrix
by mitochondrial processing peptidase (MPP) after protein
translocation across the two membranes. MPP also cleaves
off N-terminal presequences followed by hydrophobic sorting
signals in proteins localized in the inner membrane and
intermembrane space (Table 1). A hydrophobic sorting signal is
removed by additional processing peptidase, the inner membrane
protease (IMP). The N-termini of mature proteins typically starts
with stabilizing amino acids such as alanine (Ala) and serine
(Ser), according to the N-degron pathway (formerly known as
the N-end rule pathway) (van Wijk, 2015;Dissmeyer, 2019).
However, presequence processing by MPP can generate proteins
with destabilizing residues at the newly formed N-terminus. Such
FIGURE 1 | Determinants of a targeting sequence for protein import to mitochondria and chloroplasts in plant cells. N-terminal signal peptides of both mitochondrial
and chloroplast proteins contain the N-terminal specificity domain (NSD) and the C-terminal translocation domain (CTD). The NSD determines the specificity of
protein targeting, while the CTD of both targeting sequences is interchangeable. In mitochondrial presequences, the N-terminal 4-Arg motif and moderately
hydrophobic sequence motif are crucial for protein-specific import into mitochondria. In chloroplast transit peptides, the presence of a moderately hydrophobic
region in the NSD is sufficient to target a protein into chloroplasts. In the case of dual-targeted sequences (ambiguous presequences), combining the N-terminal
region of transit peptide with mitochondrial presequence results in a protein import to both mitochondria and chloroplasts. Based on Lee et al. (2019) and McKinnon
and Theg (2019). Created with BioRender.com.
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Heidorn-Czarna et al. Mitochondrial Protein Processing
destabilized protein intermediates require a second maturation
step performed by additional mitochondrial proteases such
as intermediate cleavage peptidase 55 (ICP55) or octapeptidyl
protease 1 (OCT1), also known as mitochondrial intermediate
peptidase (MIP) (Figure 2).
Mitochondrial Processing Peptidase,
MPP
Mitochondrial processing peptidase, MPP, is an ATP-
independent metallopeptidase composed of two structurally
related subunits generally designated as α-MPP and β-MPP
(Mas1 and Mas2 in S. cerevisiae, and PMPCB and PMPCA
in mammals, respectively), which interact in presequence
processing (Table 2) (Sjöling and Glaser, 1998). In Arabidopsis,
two genes encode α-MPP, and one encodes β-MPP (Millar et al.,
2001). In the MPP complex, the αsubunit is responsible for
recognizing and binding a presequence. The βsubunit, which
contains the catalytic site with an inverted Zn-binding motif
HxxEH of pitrilysin endopeptidases, performs the actual cleavage
(Dvorakova-Hola et al., 2010). Both subunits are essential for
the MPP activity (Glaser et al., 1998;Taylor et al., 2001). MPP
is a soluble, matrix-localized enzyme in yeast and mammalian
mitochondria (Figure 2B) (Hawlitschek et al., 1988;Ou et al.,
1989). In plants, the localization of MPP is unique as both
α-MPP and β-MPP subunits are integrated into the Cytochrome
b-c1 complex (complex III) of the respiratory chain as Core
proteins facing the matrix (Figure 2A) (Braun et al., 1992;
Eriksson and Glaser, 1992;Emmermann et al., 1994;Glaser et al.,
1994;Sjöling et al., 1996). In yeast, the complex III accessory
subunits Cor1 and Cor2, which show homology to β-MPP
and α-MPP, have lost the MPP enzymatic activity; however,
in mammals, the complex III preserves basal activity to only
one known substrate, the Rieske protein (Taylor et al., 2001;
Gakh et al., 2002). Studies using different plant species such
as potato (Solanum tuberosum), wheat (Triticum aestivum),
and spinach (Spinacia oleracea) showed that despite being
an integral component of Cytochrome b-c1 complex, the
processing activity of MPP is independent of electron transfer
(Emmermann et al., 1994;Braun et al., 1995;Eriksson et al.,
1996). Recently, using single-particle cryo-electron microscopy
(cryo-EM), Maldonado et al. (2021) determined the structure
and atomic model of the complex III dimer (CIII2) in the
mung bean (Vigna radiata) mitochondria. For the first time,
structural details of the MPP αand βsubunits within the plant
complex III were demonstrated. The authors showed that each
complex III monomer contains an MPP-α/βheterodimer.
Within the MPP complex, the αand βsubunits form a large
central cavity with a highly negative surface, which interacts
with positively charged presequences. The cryo-EM map
also shows specific density corresponding to Zn2+ion at
the catalytic site of the βsubunit. Furthermore, the atomic
model of plant CIII2revealed that extended N-termini of MPP
subunits span across the complex dimer and provide additional
contact to its membrane subunits (Braun, 2021;Maldonado
et al., 2021). These data strongly confirm the uniqueness of
FIGURE 2 | Intramitochondrial localization and nomenclature of proteases involved in protein processing in mammalian, yeast, and plant mitochondria. (A)
Processing proteases in plant mitochondria. Nomenclature based on the Arabidopsis thaliana mitochondrial proteolytic system. (B) Processing proteases in
mammalian and yeast mitochondria. Nomenclature written in capital letters indicates a mammalian protease, while in brackets – a yeast protease. Created with
BioRender.com.
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complex III in plants, functioning as a respiratory enzyme and a
processing peptidase.
Approximately 80% of presequences have Arg residue at
the −2 (−2R) or −3 (−3R) position relative to the mature
protein N-termini, which is surrounded by a loosely conserved
motif of R-X↓X or R-X-F/Y/L↓A/S-X, respectively (Schneider
et al., 1998;Zhang et al., 2001). The third additional group
lacks a conserved Arg (no-R) close to the maturation site;
however, in plants A. thaliana and O. sativa, there is a consensus
sequence of F/Y↓S/A for no-R group of proteins (Huang et al.,
2009). The fourth group contains Arg in position −10 (−10R)
with a consensus motif of R-X↓F/L/I-X2-T/S/G-X4↓X (Isaya
et al., 1991). The −2R, −3R, and no-R motifs were found in
presequences of all studied organisms; however, the −10R motif
is absent in plants (Zhang et al., 2001;Huang et al., 2009).
Studies have shown that the −3R motif is the most common
cleavage motif in plants, whereas, in yeast and mammals, the
most common are −2R and −3R motifs (Huang et al., 2009;
Calvo et al., 2017). The ambiguity of the presence of −2R and
−3R consensus motifs was later clarified by understanding that
the −3R motif results from a subsequent cleavage of amino acid
by ICP55 (Vögtle et al., 2009;Carrie et al., 2015;Huang et al.,
2015). Furthermore, the cleavage site in the −10R motif was later
related to the OCT1 protease (Vögtle et al., 2011).
It has long been assumed that MPP cleaves preproteins at a
specific site. Analysis of experimentally identified Arabidopsis
presequences with more than 50 amino acids, thus longer
than the average length of the A. thaliana mitochondrial
presequences (43 amino acids), showed that most of them
are likely to possess an additional MPP recognition site
(Huang et al., 2009). Indeed, the mitochondrial FTSH4 protease
precursor is cleaved by MPP at two different sites (Kmiec
et al., 2012). The two-step processing carried out by MPP
has been previously reported for yeast and human frataxin
(Branda et al., 1999;Cavadini et al., 2000) and ATP synthase
subunit 9 in Neurospora crassa (Schmidt et al., 1984). Using the
precursor of mitochondrial tandem protein Arg5,6 as a model
substrate from S. cerevisiae mitochondria, Friedl et al. (2020)
have demonstrated that MPP not only removes the N-terminal
targeting sequence from this precursor tandem protein but is
also required for specific internal processing generating two
functional enzymes, Arg5 and Arg6. Further, in silico searching
for internal matrix targeting signal-like sequences (iMTS-L) and
canonical MPP cleavage sites in mitochondrial precursor proteins
with composite structure in different species (Atp25 from
S. cerevisiae and Emericella nidulans, Etp1, Rsm22-Cox11, and
the uncharacterized SPAC22A12.08c from Schizosaccharomyces
pombe, as well as RPS14 from Oryza sativa japonica) led to
the identification of both types of motifs in these organisms
(Friedl et al., 2020). This study indicates that internal precursor
processing by MPP is conserved among fungi and plants, and
possibly other eukaryotes.
Intermediate Cleavage Peptidase of
55 kDa
Intermediate cleavage peptidase of 55 kDa (ICP55) is a highly
conserved ATP-independent metalloprotease belonging to the
aminopeptidase P family (Naamati et al., 2009;Vögtle et al.,
2009). Yeast and mammalian ICP55 orthologs are peripherally
attached to the mitochondrial inner membrane from the matrix
site (Vögtle et al., 2009). In plants, ICP55 has been found in
the soluble mitochondrial protein fraction (Migdal et al., 2017)
(Table 2 and Figure 2). Interestingly, in yeast, the isoform of
Icp55 has also been identified in the nucleus (Naamati et al.,
2009), while in humans in the cytosol (Ersahin et al., 2005;Singh
et al., 2017).
The stabilizing function of ICP55 was first shown in yeast and
human (Vögtle et al., 2009;O’Toole et al., 2010). The protease
cleaves a single destabilizing amino acid such as tyrosine (Tyr,
Y), leucine (Leu, L), and phenylalanine (Phe, F) at the −2R
consensus site (Y/L/F↓S/A) of MPP-generated N-termini, leaving
mature proteins with serine (Ser, S), alanine (Ala, A), or threonine
(Thr, T) (Vögtle et al., 2009;Teixeira and Glaser, 2013). The
analysis of the Arabidopsis mitochondrial N-terminal proteome
uncovered that plant ICP55 recognizes consensus cleavage motif
of RX(F/Y/I/L)↓(S/A)(S/T), which is remarkably similar to the
yeast and mammalian processing sites, and showed that F, Y, and
L are the most abundant amino acids removed by the Arabidopsis
ICP55 (Carrie et al., 2015;Huang et al., 2015). Notably, in
contrast to yeast, the Arabidopsis ortholog is responsible for
processing more protein substrates, i.e., approximately 52% of
Arabidopsis mitochondrial proteins are processed by ICP55,
while only 12% in S. cerevisiae (Carrie et al., 2015;Calvo et al.,
2017). Furthermore, plant ICP55 protease is also responsible for
the cleavage of non-R group proteins (Carrie et al., 2015;Huang
et al., 2015). In plants, ICP55 might also cleave some protein
substrates twice. The identification of three different N-termini
of the mitochondrial acyl carrier protein 3 led to the conclusion
that this protein is first cleaved by MPP and then twice by ICP55,
leaving the stable amino acid at the N-terminus. Yet, it is also
possible that the third cleavage is performed by some unknown
protease (Carrie et al., 2015).
The deletion of ICP55 in yeast and plants had different
biological consequences: while the icp551yeast mutants were
defective in growth on non-fermentable medium at elevated
temperature (37◦C) (Vögtle et al., 2009), the Arabidopsis icp55
T-DNA insertional lines were indistinguishable from wild-type
plants under different growth conditions (long-day and short-
day photoperiod, elevated temperature of 30, 37, or 40◦C) (Carrie
et al., 2015;Huang et al., 2015;Migdal et al., 2017). On the
other hand, in both yeast and Arabidopsis, the loss of ICP55
enhanced in vitro mitochondrial protein degradation in the
mutant compared to the wild-type (Vögtle et al., 2009;Huang
et al., 2015). The functional complementation assay showed that
the Arabidopsis ICP55 could substitute for the yeast homolog
(Migdal et al., 2017).
Octapeptidyl Aminopeptidase 1
Octapeptidyl aminopeptidase 1 (OCT1 in Arabidopsis, Oct1
in S. cerevisiae, MIP (mitochondrial intermediate peptidase
in mammals) is, like MPP and ICP55, an ATP-independent
metalloprotease, which was first characterized in rat liver
mitochondria and initially named as matrix processing protease
II to distinguish it from MPP (Kalousek et al., 1988). In yeast
and mammals, the protease is a soluble protein located in the
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mitochondrial matrix (Isaya et al., 1992;Kalousek et al., 1992),
while in plants, it is a membrane-bound enzyme (Table 2 and
Figure 2) (Migdal et al., 2017). Oct1 cleaves eight amino acids
(octapeptides) from the MPP-generated N-terminal sequences in
the protein substrates, which contain Arg in the −10 position
from the MPP recognition site (Isaya et al., 1992;Vögtle et al.,
2011). Interestingly, the presence of Arg in position −10 of
Oct1 substrates is not always a strict requirement as some
precursor proteins contain lysine (Prx1, Peroxiredoxin 1) or
cysteine, alanine, or aspartate (Imo32, Intermediate cleaved by
mitochondrial octapeptidyl aminopeptidase 1) at this position
(Vögtle et al., 2011). In plants, mitochondrial presequences lack
the −10R motif (Huang et al., 2009), and the identified small
set of OCT1 substrates in the Arabidopsis mitochondria did not
show the presence of that motif (Carrie et al., 2015). Interestingly,
Carrie et al. (2015) also indicated that Arabidopsis OCT1, aside
from its processing activity following MPP, can also process
mitochondrial presequences without the prior cleavage by MPP.
The in vitro import into Arabidopsis mitochondria has shown
that the B13 subunit of complex I is cleaved exclusively by
OCT1 after its insertion into complex I (Carrie et al., 2015).
The Arabidopsis plants lacking OCT1 protease did not show
any phenotypic and mitochondrial respiratory activity alterations
(Carrie et al., 2015;Migdal et al., 2017), in contrast to the
oct11yeast mutant, which was respiratory deficient when grown
on a non-fermentable carbon source (Isaya et al., 1994). The
functional complementation experiment showed that the plant
homolog of Oct1 could not restore respiratory function in the
oct11yeast mutant suggesting no conservation between the yeast
and plant Oct1 proteases (Migdal et al., 2017).
Inner Membrane Protease
Mitochondrial precursor proteins destined to the IMS or
facing the IMS often contain the hydrophobic sorting signal,
which is cleaved off by the mitochondrial inner membrane
protease (IMP). IMP is an integral protein embedded in the
inner membrane, with its catalytic C-terminus facing the IMS
(Figure 2) (Nunnari et al., 1993). The enzyme can process both
nuclear and mitochondrial encoded proteins, and the cleavage
is usually preceded by the processing of a substrate protein by
MPP (Mossmann et al., 2012). Recent studies by Gomes et al.
(2017) demonstrated that in yeast, proteolytic maturation of Prx1
involves three proteases, MPP, Oct1, and Imp, which control its
submitochondrial localization.
Inner membrane protease has been so far primarily studied in
yeast S. cerevisiae, in which it exists as a heterodimeric complex
consisting of two catalytic subunits, Imp1 and Imp2, and the
auxiliary protein Som1, required for the catalytic activity of Imp1
(Schneider et al., 1991;Nunnari et al., 1993;Esser et al., 1996).
Imp1 and Imp2 belong to the same serine protease family, yet
they recognize a distinct set of substrates (Table 2) (Nunnari et al.,
1993). The repertoire of known protein substrates of Imp1 and
Imp2 is relatively small. Only seven protein substrates of Imp1
have been identified in yeast mitochondria, with Mcp3 protein
lately reported as the first known mitochondrial outer membrane
protein processed by Imp1 protease (Table 2) (Sinzel et al.,
2016). Interestingly, Imp1 not only processes N-terminal sorting
signals but can also cleave off the C-termini. For example, the
enzyme removes the C-terminal targeting sequence of the TIM23
subunit Mgr2 and promotes proper assembly of the complex
(Ieva et al., 2013).
For the moment, there are only two known Imp2 substrates,
cytochrome c1 (cyt c1, subunit of complex III) and Prx1 (Table 2).
Sorting of Prx1 into the IMS requires the cleavage of the protein
by Imp2 alone. Alternatively, the localization of Prx1 into the
mitochondrial matrix requires the cleavage by MPP followed by
the processing by Oct1 protease (Gomes et al., 2017).
Mammalian IMP proteases (in human IMMP1L and
IMMP2L, Table 2) have been studied mainly by expression in
yeast. So far, there are only three known IMP substrates identified
in vitro, such as Smac/DIABLO cleaved by the mouse IMP1
(Burri et al., 2005), as well as cyt c1 and an ortholog of yeast Gut2,
FAD-dependent glycerol-3-phosphate dehydrogenase cleaved by
IMP2 in the mouse mitochondria (Table 2) (Luo et al., 2006). Cyt
c1 is a proteolytic substrate for the yeast Imp2 and mammalian
IMP2; on the other hand, glycerol-3-phosphate dehydrogenase
has switched from Imp1 in yeast to IMP2 in mammals.
Comparative sequence analyses of the yeast and plant
proteolytic system components revealed that the Arabidopsis
genome contains six putative Imp homologs (Kwasniak et al.,
2012), and among them, two have been analyzed (IMP1a,
At1g53530; IMP2, At2g31140) (Migdal et al., 2017). Both IMP1a
and IMP2 showed mitochondrial localization, and IMP1a has
been found in the membrane fraction, similarly to the yeast
counterpart (Figure 2). The morphology, development, and
mitochondrial respiration of the Arabidopsis imp1a T-DNA
insertional line was comparable to the wild-type plants (Migdal
et al., 2017). On the contrary, yeast imp11and imp21
knockout mutants showed respiratory growth defects on the
non-fermentable carbon sources (Burri et al., 2005). However,
the Arabidopsis IMP1a and IMP2 orthologs could not restore
the respiratory defects in the functional complementation assay,
implying a functional divergence of the yeast and plant IMP
(Migdal et al., 2017). No physiological plant IMP substrates have
been identified to date.
MITOCHONDRIAL PROTEASES WITH
ADDITIONAL FUNCTION OF LIMITED
PROTEOLYSIS
ATP23 Protease
The functional analysis of ATP23 metalloprotease has been
primarily performed in yeast. The protease, which localizes to
the IMS, shows diverse functions ranging from precursor protein
processing and chaperone activity to protein turnover control
(Mossmann et al., 2012). In the S. cerevisiae mitochondria,
Atp23 mediates the proteolytic cleavage of the presequence of
Atp6, the mitochondrially encoded subunit of ATP synthase,
after its insertion into the inner membrane (Osman et al.,
2007;Zeng et al., 2007). Atp23, together with Atp10, also
works as a chaperone in the correct assembly of Atp6 into the
ATP synthase complex (Osman et al., 2007;Zeng et al., 2007).
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Furthermore, Atp23 participates in the degradation of Ups1,
the IMS protein involved in regulating the distribution of
cardiolipin and phosphatidylethanolamine in the mitochondrial
inner membrane (Potting et al., 2010) (Table 2).
The function of ATP23 in plant and mammalian mitochondria
is still unknown. The mammalian Atp6 ortholog is synthesized
without the N-terminal targeting signal characteristic for the
yeast Atp6, indicating that in mammals, this protein is not under
the control of ATP23 (Zeng et al., 2007). The Arabidopsis ATP23
has been found in the soluble mitochondrial protein fraction,
but the experiments in obtaining T-DNA insertional lines in the
ATP23 gene were unsuccessful (Migdal et al., 2017). In yeast, the
deletion of the ATP23 gene results in respiratory deficiency when
the cells are grown on a non-fermentable carbon source (Osman
et al., 2007;Zeng et al., 2007). However, the complementation of
the atp231yeast mutants with the Arabidopsis ATP23 protein
did not restore yeast respiratory function, and the plant ATP23
could not process yeast Atp6 (Migdal et al., 2017). These findings
raise the possibility that ATP23 acts on different substrates in
plants and mammals.
Rhomboid Protease
Rhomboids are integral membrane ATP-independent serine
proteases. This class of proteases is fascinating since it is involved
in specific proteolysis, known as regulated intramembrane
proteolysis (RIP), in a membrane environment. RIP is a
mechanism by which a membrane-anchored protein substrate
is cleaved at its transmembrane region by a rhomboid protease
within the lipid bilayer and further released as an active protein
(Wolfe and Kopan, 2004). Members of this protease family have
been shown to localize to mitochondria in yeast, plants, and
mammals (Table 2) (McQuibban et al., 2003, 2006;van der Bliek
and Koehler, 2003;Kmiec-Wisniewska et al., 2008;Adamiec et al.,
2017).
In yeast S. cerevisiae, the mitochondrial rhomboid protease
Pcp1 (Processing of cytochrome cperoxidase) is involved
in proteolytic cleavage of two substrates, the cytochrome c
peroxidase (Ccp1) (Esser et al., 2002) and mitochondrial genome
maintenance 1 (Mgm1) (Table 2) (Herlan et al., 2003;Schäfer
et al., 2010). Besides being cleaved by Pcp1, the proteolytic
maturation of Ccp1 and Mgm1 requires the activity of additional
proteases. Before cleavage by Pcp1, Ccp1 is integrated into the
inner membrane as a precursor protein through the chaperone
activity of the m-AAA protease (Schäfer et al., 2010). The second
substrate, Mgm1, first undergoes processing by MPP, followed by
further proteolytic cleavage by Pcp1 (Herlan et al., 2003).
Interestingly, the mammalian ortholog of the yeast Pcp1,
PARL (presenilin-associated rhomboid-like) protease, does not
mediate processing of the Mgm1 homolog OPA1 (optic atrophy
1) (Duvezin-Caubet et al., 2007;Anand et al., 2014). Both Mgm1
and OPA1 are GTPases involved in the mitochondrial fusion
and cristae formation; however, in mammals, the proteolytic
processing of OPA1 is controlled by two other inner membrane
proteases, the i-AAA YME1L and OMA1 (Griparic et al.,
2007;Head et al., 2009;Anand et al., 2014). Instead, PARL
cleaves two proteins known to play a role in mitophagy, kinase
PINK1 (phosphatase and tensin (PTEN)-induced kinase 1)
(Jin et al., 2010;Meissner et al., 2011) and phosphatase PGAM5
(phosphoglycerate mutase family member 5) (Sekine et al., 2012).
A recent in vitro study reported that PARL is also required for
the cleavage of TTC19, a subunit of complex III, and the pro-
apoptotic protein Smac-DIABLO (Table 2) (Saita et al., 2017).
Impaired proteolytic maturation and expression of TTC19 in the
mitochondria of Parl−/−mouse brain tissue has been recently
demonstrated (Spinazzi et al., 2019).
Knowledge concerning the function of mitochondrial
rhomboid proteases in plants is still limited. Out of four
predicted as mitochondrially targeted rhomboid-like proteases in
A. thaliana (Adamiec et al., 2017), only one ortholog of the yeast
Pcp1, the RBL12 rhomboid protease, has been experimentally
characterized in plant mitochondria (Kmiec-Wisniewska et al.,
2008). The mitochondrial localization of the RBL6, RBL15,
and RBL16 rhomboid homologs needs to be verified (Adamiec
et al., 2017). The complementation of the yeast pcp11mutants
with the Arabidopsis RBL12 open reading frame indicated
that the plant ortholog does not process the yeast substrates,
Ccp1 and Mgm1. These results imply that despite the high
similarity at the amino acid sequence level between the yeast and
Arabidopsis mitochondrial rhomboids, the substrate recognition
and processing mechanism by plant RBLs evolved differently
(Kmiec-Wisniewska et al., 2008). It is especially interesting
since yeast substrates were processed in vitro by the mammalian
ortholog, PARL protease (McQuibban et al., 2003).
In plant mitochondria, inner membrane carrier proteins
possess an N-terminal extension removed in a two-step
processing upon import into the organelle (Murcha et al., 2004).
It has been demonstrated that the first cleavage is performed by
MPP, while the second processing probably occurs in the IMS and
is carried out by an undefined yet serine protease. The authors
proposed that a rhomboid protease may play a role in the second
cleavage (Murcha et al., 2004).
Mitochondrial Inner Membrane
Metallopeptidases: ATPase Associated
With Various Cellular Activities Proteases
and OMA1
Two highly conserved hexameric AAA (ATPase associated with
various cellular activities) proteases, i-AAA and m-AAA, have
been found in the mitochondrial inner membrane of virtually all
eukaryotes. The i-AAA exposes its catalytic center to the IMS,
whereas m-AAA is directed toward the mitochondrial matrix.
Both AAA proteases contain an AAA domain and the proteolytic
domain with a conserved Zn2+-binding motif (HExxH) within
a single subunit. The AAA domain, which has a chaperone-like
activity, binds and hydrolyzes ATP and delivers substrates to the
proteolytic center (Glynn, 2017;Opalinska and Janska, 2018).
In yeast, the m-AAA proteases are organized as hexameric
complexes composed of the Yta12 and Yta10 subunits (Arlt et al.,
1996). The mammalian m-AAA forms either homo-oligomeric
complexes consisting of AFG3L2 subunits only or hetero-
oligomeric complexes composed of AFG3L2 and paraplegin (also
known as SPG7) (Koppen et al., 2007). Similarly, plant m-AAA
complexes are composed of FTSH3 and FTSH10 subunits that
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can form either homo- or hetero-oligomers (Piechota et al.,
2010). In contrast, the i-AAA protease is built from six subunits
of Yme1 in yeast and YME1L in mammals (Weber et al.,
1996). There is also only one i-AAA protease in plants, FTSH4
(Table 2 and Figure 2) (Gibala et al., 2009;Smakowska et al.,
2016). Earlier studies reported the presence of another i-AAA
complex, FTSH11, in the mitochondria and chloroplasts of
plants grown under short-day photoperiod (Urantowka et al.,
2005); however, recent proteomic analyses demonstrated that
FTSH11 is exclusively localized to the chloroplasts (Adam
et al., 2019), at least when plants were grown under long-day
conditions for 4 weeks.
The i-AAA and m-AAA proteases play a major role in the
general quality control of mitochondrial proteome (mtPQC,
mitochondrial protein quality control). The proteases remove
misfolded, mislocalized, non-assembled, or damaged proteins
by breaking them into small peptide fragments (Leonhard
et al., 2000). This rapid degradation prevents the formation of
potentially toxic protein aggregates within the organelle (Maziak
et al., 2021). Aside from their critical role in the mtPQC,
the i-AAA and m-AAA proteases also perform highly specific
proteolytic reactions by controlling the life span of regulatory
proteins involved in key metabolic pathways (Quiros et al.,
2015). Generally, the substrate spectrum of i-AAA and m-AAA is
relatively broad as the proteases degrade not only the IM protein
substrates but also the OM and IMS (i-AAA), as well as the matrix
(m-AAA) localized proteins (Opalinska and Janska, 2018;Song
et al., 2021).
In recent years, several studies have reported the role of
i-AAA and m-AAA in limited proteolysis. One of the best-
known examples is the maturation of MrpL32 protein, a nuclear-
encoded subunit of the mitochondrial ribosome, which requires
cleavage of the N-terminal presequence by the m-AAA protease
(Table 2). This process is crucial for ribosomal biogenesis
and, in consequence, mitochondrial translation and OXPHOS
formation. In yeast, the processing of MrpL32 is postulated to
be a central function of the m-AAA protease as the synthesis of
mitochondrially encoded proteins was strongly impaired in the
cells lacking m-AAA, and the cells were respiratory incompetent
(Nolden et al., 2005). Similarly, a decreased rate of mitochondrial
protein synthesis and defective ribosome assembly associated
with the lack of MrpL32 processing have also been observed in
the AFG3L2 or paraplegin-deficient mammalian mitochondria
(Nolden et al., 2005;Almajan et al., 2012). Lately, Kolodziejczak
et al. (2018) have shown that the Arabidopsis m-AAA proteases,
FTSH3 and FTSH10, are able to process plant mitochondrial
AtL32 protein, a homolog of the yeast and mammalian MrpL32
(Table 2). In addition, in organello protein synthesis revealed that
the translation of mitochondrially encoded proteins was strongly
impaired in the Arabidopsis mutant lacking both FTSH3 and
FTSH10 proteases, but not in plants lacking one of the m-AAA.
Taken together, the specific processing activity of m-AAA toward
the ribosomal protein is conserved across diverse eukaryotes,
including yeast, mammals, and plants. The processing activity
of the m-AAA protease has also been shown toward another
substrate, the mitochondrial protease OMA1. In mammals, the
m-AAA AFG3L2 subunit is involved in the proteolytic processing
of the 60-kDa precursor form of OMA1 to its mature 40-kDa
protein (Consolato et al., 2018).
The question is, how does the m-AAA protease distinguish
partial processing from a complete protein degradation? It has
been shown for MrpL32 that the m-AAA protease does not
recognize a specific cleavage site. Instead, it starts degrading a
protein from its N-terminal unstructured end until reaching a
tightly folded C-terminal part of MrpL32 harboring a conserved
Cys-rich zinc-binding motif (Bonn et al., 2011). The formation
of a folded domain terminates the degradation of a protein
and induces the release of mature MrpL32. Plant mitochondrial
ribosomal protein AtL32, similarly to yeast MrpL32, contains
a Cys-rich sequence motif essential for ribosomal protein
processing (Kolodziejczak et al., 2018). It is also possible that
partial processing of other mitochondrial protein substrates
occurs through a mechanism similar to MrpL32.
Another example of limited proteolysis includes Atg32
protein, the mitochondrial OM-localized receptor involved in
tagging mitochondria for autophagosomal degradation in yeast
(Okamoto et al., 2009). The C-terminal domain of the protein
is exposed into the IMS, where it is cleaved by the yeast i-AAA
protease, Yme1 (Table 2) (Wang et al., 2013). This process
generates the mature form of Atg32 that directly interacts
with Atg11 and Atg8 proteins, leading to the formation of
mitophagosome (Okamoto et al., 2009). There is no close
Atg32 homolog in A. thaliana, and no proteins on the plant
mitochondrial outer membrane with similar function have been
experimentally confirmed to date. Recently, Broda et al. (2018)
used a bioinformatic tool to identify in silico the Arabidopsis
mitochondrial proteins that are specifically recognized by the
Atg8 protein, a central protein of the autophagy machinery, based
on the presence of a short Atg8-interacting motif (AIM) (Xie
et al., 2016). Among the AIM-containing proteins, there are outer
membrane proteins, which may play the role of mitochondrial
receptors in the activation of mitophagy (Broda et al., 2018).
It can be assumed that similarly to yeast, the Arabidopsis
Yme1 ortholog, FTSH4 protease, activates mitophagy through
proteolytic cleavage of specific OM-located substrates, but this
needs to be confirmed. So far, a few proteins have been identified
as proteolytic substrates of FTSH4, but there is no experimental
evidence about the processing activity of this enzyme in plants
(Opalinska et al., 2017, 2018;Maziak et al., 2021).
OMA1 protease has been initially identified in yeast as the
mitochondrial inner membrane protease capable of replacing
m-AAA in the proteolytic degradation of Oxa1 protein (hence
original name overlapping with the m-AAA protease 1) (Käser
et al., 2003). The homologs of OMA1 have been found in
both prokaryotes and eukaryotes, with some exceptions, such
as Drosophilidae, Nematoda, and Trematoda (Levytskyy et al.,
2017). OMA1 is a metalloprotease containing a conserved Zn2+-
binding motif (HExxH), but unlike the i-AAA and m-AAA
proteases, it lacks the AAA domain and thus is ATP-independent.
In yeast and mammals, the enzyme is mainly dormant under
normal conditions; however, heat and oxidative stress and the
loss of mitochondrial inner membrane potential lead to rapid
activation of OMA1 (Baker et al., 2014;Bohovych et al., 2014;
Zhang et al., 2014). It has been reported that the yeast Oma1
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mediates degradation of newly synthesized, non-assembled Cox1
in cells lacking the Coa2 assembly factor (Khalimonchuk et al.,
2012). The functional complementation assay demonstrated that
the Arabidopsis OMA1 could partially replace the degradation
function of the yeast protease (Migdal et al., 2017). There is
no knowledge about the Oma1 processing activity in yeast to
date. In addition to its significant role in the maintenance of
mitochondrial bioenergetics, ultrastructure, and the stability of
respiratory complexes in yeast, mammals, and plants (Bohovych
et al., 2015;Migdal et al., 2017;Viana et al., 2021), OMA1 has
been well recognized as a significant player, which together with
the i-AAA protease controls the processing of mitochondrial
dynamics regulator OPA1 in mammals (Table 2) (Griparic et al.,
2007;Ehses et al., 2009;Head et al., 2009;Anand et al., 2014).
As stated earlier, OPA1, and its yeast homolog Mgm1, are small
GTPases that mediate mitochondrial fusion. In yeast, however,
Mgm1 is proteolytically cleaved by rhomboid protease Pcp1
(van der Bliek and Koehler, 2003).
Generally, in mammals, OPA1 is present in different isoforms
known as long OPA1 (L-OPA1), which represents an unprocessed
form, and short OPA1 (S-OPA1) obtained by the proteolytic
cleavage at two different cleavage sites, S1 and S2, by OMA1 and
YME1L proteases, respectively (Anand et al., 2014). It has been
displayed that the cleavage at the S1 is performed at the position
R194-A195, while at the S2 - within the 217LQQQIQE223
sequence (Ishihara et al., 2006). Under non-stress conditions,
YME1L mediates the processing of some of the L-OPA1 isoforms
giving relatively equal amounts of L-OPA1 and S-OPA1, which
is required to maintain the balance between mitochondrial
fusion and fission. Under metabolic changes or mitochondrial
dysfunction, the fission of mitochondrial inner membrane is
triggered by activation of OMA1, which mediates rapid cleavage
of all L-OPA1 isoforms into the short variants leading to
the subsequent fragmentation of the mitochondrial network.
This process enables the segregation and removal of damaged
mitochondria through mitophagy (Anand et al., 2014). On the
other hand, impaired activity of OMA1 prevents mitophagy by
stabilizing L-OPA1, thus hindering mitochondrial fragmentation
(MacVicar and Lane, 2014). Furthermore, OMA1 may also
regulate apoptosis by mediating OPA1 proteolysis, leading to
mitochondrial fission and leakage of proapoptotic factors in
ATP-depleted renal tubular cells (Xiao et al., 2014).
Recently, Guo et al. (2020) have identified OMA1 as one of
the molecular components of the integrated stress response (ISR)
pathway in mammalian cells that signals mitochondrial stress
to the cytosol. The ISR involves OMA1-dependent cleavage of
the mitochondrial inner membrane protein DELE1 to its shorter
form (DELE1s), which accumulates in the cytosol and triggers the
stress response. The cleavage of DELE1 occurs at histidine 142
and does not require a specific sequence motif (Guo et al., 2020).
Lately, mitochondrial small heat-shock proteins (sHSPs) have
been found to be under proteolytic control of the Arabidopsis
OMA1 and FTSH4 proteases when plants are grown under
prolonged moderate heat stress (Maziak et al., 2021). Under these
conditions, sHSPs, identified in insoluble mitochondrial protein
aggregates, were subjected to complete degradation rather than
limited proteolysis as shown by the in vitro degradation assay
in the Arabidopsis mitochondria in the presence of externally
added OMA1 or FTSH4 synthesized in the cell-free expression
system. The OMA1- and FTSH4-dependent proteolytic control
of sHSPs appears to be unique to plants since organelle-targeted
small heat-shock proteins have been so far identified in plants
only, with an exception for a mitochondrion-targeted sHSP22
in Drosophila melanogaster (Maziak et al., 2021). There is no
knowledge about the OMA1 processing activity to date in plants.
METHODS FOR CHARACTERIZATION
OF MITOCHONDRIAL PROTEIN
N-TERMINI, PROTEASE RECOGNITION
MOTIF, AND PROTEOLYTIC
SUBSTRATES
Proteolytic processing produces two polypeptide chains where
one possesses a protease-generated neo-N-terminus and the
second a neo-C-terminus. Generated protein termini provide
precise information about a protease cleavage site and specify
a recognition motif within the protein substrates. The current
proteomic methods to study native and neo termini of proteins
(i.e., before and after proteolytic cleavage) are mass-spectrometry
(MS) based N- and C-terminomics techniques, which facilitate
identification of the N- and C-terminomes from the total
proteomes. These techniques mostly rely on the bottom-up
proteomics, in which a site-specific externally added protease
digests proteomes to generate peptides before chromatographic
separation and tandem MS (MS/MS) analysis, in contrast to
the top-down proteomics approach, which directly analyzes
intact proteins with all retained modifications by MS (Aebersold
and Mann, 2016). Various proteomics technologies for N-
and C-terminal analysis have been recently comprehensively
described (reviewed in Tanco et al., 2015;Demir et al., 2018;
Kaleja et al., 2019;Luo et al., 2019;Perrar et al., 2019;
Bogaert and Gevaert, 2020;Kaushal and Lee, 2021;Mintoo
et al., 2021). Due to some technical difficulties (e.g., poor
ionization efficiencies of protein C-termini, low yielding carboxyl
group labeling strategies), C-terminomics technologies are,
however, less successfully implemented and widely used, in
comparison to the N-terminomics methods, which in recent
years have become an indispensable tool in positional proteomics
(Eckhard et al., 2016).
Historically, the study of N-terminal protein sequences has
been performed using the Edman degradation method, also
known as N-terminal protein sequencing (Edman et al., 1950).
For example, this method has been applied to determine
the N-termini of potato tuber and Arabidopsis mitochondrial
proteins (Jänsch et al., 1996;Millar et al., 1998;Kruft
et al., 2001). Because of its limitations in automatization and
inability to sequence N-terminally labeled proteins, the Edman
degradation method became impractical for the N-termini
characterization and was replaced by high-throughput MS-based
N-terminomics technologies.
Generally, in N-terminomics, the strategies for selective
isolation of N-terminal peptides from other digested internal
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peptides rely on two critical objectives. First, they utilize the
unique reactivity of the primary amine at the protein N terminus,
making the amine labeling highly favorable, in contrast to the
less reactive carboxyl group (Mintoo et al., 2021). Second, they
depend on selective enrichment of labeled N-terminal peptides by
employing either a positive or negative selection approach, which
reduces the complexity of a peptide sample. A positive selection
strategy relies on a direct enrichment of N-terminal peptides
from the peptide mixture [e.g., the Subtiligase and Chemical
Enrichment of Protease Substrates (CHOPS) methods]. In the
negative selection methods, the N-terminal peptides are enriched
indirectly by the removal of undesired (internal, C-terminal)
peptides (e.g., COFRADIC, ChaFRADIC, and TAILS methods)
(Bogaert and Gevaert, 2020).
This chapter provides a short overview of a few
N-terminomics techniques based on the negative selection
approach, COFRADIC, ChaFRADIC, and TAILS, with their
strengths and limitations. These methods have been successfully
applied to generate global profiling of cellular and organellar
N-terminomes, describe protease cleavage patterns, and
identify proteolytic substrates in yeast, mammalian, and plant
mitochondria (Figure 3).
Combined Fractional Diagonal
Chromatography
The N-terminal COFRADIC (combined fractional diagonal
chromatography) is one of the earlier bottom-up proteomics
technologies to enrich the N-terminal peptides. COFRADIC was
initially developed by Gevaert et al. (2003), who introduced the
concept of diagonal reversed-phase (RP) chromatography for
isolating protein N-termini by depleting the internal peptides.
The general principle of COFRADIC is as follows. After isolation
and denaturation of studied proteomes, cysteine residues are
first reduced and alkylated. This sample preparation step is
commonly used in many, if not all, N-terminomics studies
(Bogaert and Gevaert, 2020). Subsequently, all primary amines
[α- and ε-amines of protein N-terminus and lysine (Lys) residues]
are labeled using N-hydroxysuccinimide (NHS) acetate ester
in N-acylation reaction. At this stage, it is possible to employ
differential stable isotopes of NHS to mass-tag native and neo-
N-terminal peptides allowing the distinction between different
protein samples (e.g., protease-inactive mutant versus wild-
type) (Staes et al., 2011) (Figure 3A). The differentially labeled
samples are pooled and digested by trypsin together. Typically,
trypsin cleaves peptide bonds at the C-terminal end of Arg and
Lys residues; however, here, the digestion generates only Arg-
ending peptides, as trypsin does not recognize acetylated lysine
residues. The obtained peptides undergo the first separation by
reversed-phase high-performance liquid chromatography (RP-
HPLC), in which each peptide elutes after a particular retention
time (RT) (Figure 3B). All collected peptides are then reacted
with 2,4,6-trinitrobenzenesulfonic acid (TNBS). In this reaction,
only internal and C-terminal peptides will be blocked because
of the formation of a very hydrophobic 2,4,6-trinitrophenyl
(TNP) group at their free α-amines. Each peptide fraction is
then run on the second RP-HPLC using identical conditions
as the primary run. However, since the strong hydrophobicity
of the TNP group leads to a shift (an increase) in retention
time of the TNBS-modified peptides, they elute later than in a
primary run. In contrast, the peptides with labeled native and
neo-N-termini have unaltered hydrophobicity and elute within
the same time intervals as in the primary HPLC run. The native
and neo-N-terminal peptides are collected and identified by
liquid chromatography-tandem mass spectrometry (LC-MS/MS)
(Figure 3B), providing the identity of protease substrates and
protease cleavage sites (Staes et al., 2011;Luo et al., 2019;Mintoo
et al., 2021).
So far, COFRADIC has been used in a broad spectrum
of studies in different organisms. For example, this technique
has been employed to identify physiological substrates of
metacaspase 9 (MC9) in A. thaliana seedlings (Tsiatsiani et al.,
2013) as well as for the identification, in combination with
ribosome profiling, of novel translation initiation sites in
Arabidopsis (Willems et al., 2017). This technique has also been
employed to characterize protein substrates of HhoA, HhoB, and
HtrA proteases in cyanobacterium Synechocystis sp. PCC 6803
(Tam et al., 2015). In humans, COFRADIC has been used to
determine cleavage specificity and substrate identities of caspases
(Van Damme et al., 2005;Wejda et al., 2012) and HIV-1 protease
(Impens et al., 2012).
Notably, the COFRADIC method has been applied in
the global analysis of the yeast S. cerevisiae mitochondrial
N-terminome (Vögtle et al., 2009). In this landmark study, the
N-termini of 615 mitochondrial proteins has been identified.
Some mitochondrial proteins exhibited two N-termini differing
by one amino acid residue. This observation led to identifying the
Icp55 peptidase responsible for cleaving a single amino acid from
−2R presequences following MPP cleavage, converting unstable
intermediates into stable proteins. This study has also solved
the controversial problem of the two MPP cleavage motifs (−2R
and −3R), revealing that the −3R does not constitute an MPP
cleavage site motif. Instead, it is a part of a motif for the two-step
cleavage, first by MPP and then by Icp55 (Vögtle et al., 2009).
Furthermore, in this work, new protein substrates of another
processing protease, Oct1, have also been identified (Vögtle
et al., 2009). In a follow-up study, a comprehensive analysis
of the N-termini of the Oct1 substrate intermediates compared
to mature proteins revealed the presence of a destabilizing
N-terminal amino acid in the intermediate form. This finding
has uncovered that the processing by Oct1 converts unstable
precursor intermediates generated by MPP into stable proteins
(Vögtle et al., 2011).
To date, there is no information on the use of COFRADIC
in identifying protein N-termini and processing substrates in
plant mitochondria.
Since its original publication, some critical improvements
to the COFRADIC protocol have been made. It has been
observed that, after digestion, internal peptides containing the
N-terminal glutamine residues may form spontaneously cyclic
N-pyroglutamyl peptides, which do not react with TNBS and thus
cannot be removed in the follow-up sorting process. To prevent
it, two enzymatic reactions with glutamine cyclotransferase
and pyroglutamyl aminopeptidase have been introduced, which
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Heidorn-Czarna et al. Mitochondrial Protein Processing
FIGURE 3 | Scheme of the N-terminomics workflow for identifying a protease cleavage site and processing substrates using COFRADIC, ChaFRADIC, and TAILS as
negative selection approaches for isolating N-terminal peptides in mitochondrial proteome studies. (A) Mitochondrial proteins isolated from the control and
protease-deficient plants (containing protein neo-N- and native N-termini, respectively) are subjected to labeling all primary (αand ε) amines using stable differential
isotopes to mass-tag native and neo-N-terminal peptides. Samples are pooled and digested by trypsin together. As a result, new primary α-amines at the N-termini
of internal and C-terminal peptides are generated. (B) In COFRADIC and ChaFRADIC, the obtained peptides undergo the first separation by reversed-phase
high-performance liquid chromatography (RP-HPLC) (COFRADIC) or by strong cation exchange (SCX) chromatography (ChaFRADIC). All collected peptides are
modified with TNBS (in COFRADIC) or d3-NHS (in ChaFRADIC) to alter the retention time of internal and C-terminal peptides. The peptides are then subjected to the
second identical chromatography step, in which only previously labeled N-terminal peptides are collected. In the case of TAILS, tryptic peptides are incubated with a
polymer HPG-ALD. In this reaction, only internal peptides will bind to the polymer. The polymer-bound internal peptides are removed by centrifugation, while the
N-terminal peptides are further recovered by filtration. In each type of experiment, the N-terminal peptides are analyzed by LC-MS/MS, which provides the protease
cleavage site and identifies processing protein substrates. COFRADIC, combined fractional diagonal chromatography; ChaFRADIC, charge-based fractional
diagonal chromatography; TAILS, terminal amine isotope labeling of substrates; TNBS, 2,4,6-trinitrobenzenesulfonic acid; d3-NHS, trideutero N-hydroxysuccinimide;
HPG-ALD, high molecular weight polyglycerol aldehyde polymer; LC-MS/MS, liquid chromatography-tandem mass spectrometry. Details regarding specific
methodologies are described in the main text. Based on Demir et al. (2018). Created with BioRender.com.
eliminate pyroglutamate residues and expose a new α-primary
amine for the TNBS reaction (Staes et al., 2008). Furthermore,
an additional pre-enrichment step based on the strong cation
exchange (SCX) chromatography at a pH of 3 for blocked
(e.g., acetylated) N-terminal peptides has been introduced before
the first RP-HPLC runs (Staes et al., 2008). Finally, an extra
oxidation step of methionine with hydrogen peroxide (H2O2)
has been included between the primary and secondary RP-HPLC
separations and after TNBS modification. This step causes a
shift in the retention time to an earlier point of the N-terminal
methionine-containing peptides, segregating them from the
non-methionine-containing N-terminal peptides (Gevaert et al.,
2002;Van Damme et al., 2009). Despite being successfully
employed in a wide range of applications, COFRADIC also has
some limitations, which include the requirements for specific
instruments and significant expertise as well as a relatively large
amount of protein as a starting material (1–3 mg) (Perrar et al.,
2019;Mintoo et al., 2021).
Charge-Based Fractional Diagonal
Chromatography
Charge-based fractional diagonal chromatography is a modified
version of COFRADIC, which has been developed by the group
of Venne et al. (2013) and for the first time implemented
in the studies of the yeast mitochondrial N-terminome. This
method utilizes the altered charged state of the tryptic peptides
to separate native and neo-N-terminal peptides from the peptide
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Heidorn-Czarna et al. Mitochondrial Protein Processing
mixture rather than their modified hydrophobicity. In addition,
peptides are fractionated by SCX chromatography instead of RP-
HPLC used in COFRADIC (Figure 3B). To label all primary
amines, the isolated and then reduced proteins are dimethylated
using formaldehyde. At this point, the incorporation of stable
isotope dimethyl labeling (e.g., heavy and light dimethyl labels)
enables quantitative differential analysis of two types of samples
(Figure 3A). After pooling the samples and digesting them
by trypsin, which hydrolyzes the peptide bonds C-terminal
of Arg only, the peptide mixtures are separated by first SCX
chromatography run at a pH of 2.7 (Figure 3B). After the first
SCX run, which allows for selective separation of charged peptide
fractions +1, +2, +3, +4, and >+4, all fractions are collected and
treated with trideutero N-hydroxysuccinimide (d3-NHS) acetate
(Venne et al., 2013;Bogaert and Gevaert, 2020). Only internal
and C-terminal peptides with free α-amines are acetylated in
this reaction, while the N-terminal peptides with blocked (i.e.,
labeled) α-amines remain unchanged. Since acetylation alters
the charge of internal and C-terminal peptides, these modified
peptides elute earlier in the second SCX chromatography run
than during the first separation. The retention time of N-terminal
peptides remains the same, allowing selective collection of
native and neo-N-terminal peptides (Bogaert and Gevaert, 2020;
Mintoo et al., 2021). The follow-up LC-MS/MS analysis provides
the protease cleavage site and identifies processing protein
substrates (Figure 3B).
Although ChaFRADIC, like COFRADIC, requires specific
instruments and expertise, it is generally less labor-intensive.
However, the main advantages, such as reductions in the amount
of starting material (50–200 µg) and LC-MS/MS operation time,
make ChaFRADIC more cost-effective than COFRADIC (Perrar
et al., 2019;Mintoo et al., 2021). Additional improvements have
been implemented in the past years, increasing this strategy’s
power, sensitivity, and reproducibility. For example, Venne et al.
(2015) have extended ChaFRADIC workflow by iTRAQ (Isobaric
Tags for Relative and Absolute Quantitation) labeling and multi-
enzyme protein digestion with trypsin, GluC, and subtilisin
to analyze the N-terminal proteome of A. thaliana seedlings.
This approach led to the identification of novel N-termini with
increased overall proteome coverage. Recently, further reduction
in the amount of a starting material (less than 5 µg of protein per
condition) has been introduced in a tip-version of ChaFRADIC
(ChaFRAtip) by a replacement of the SCX columns with the
pipette tips containing SCX resins (Shema et al., 2018).
As stated earlier, for the first time, ChaFRADIC has been
successfully applied in a study of the yeast mitochondrial protein
N-termini (Venne et al., 2013). This pioneering work has enabled
the differential quantitation of 1,459 non-redundant N-terminal
peptides between two S. cerevisiae protein samples within a
much shorter time and only 50 µg of mitochondrial proteins
as a starting material. Furthermore, in this work, a quantitative
comparative analysis of the yeast wild-type and icp551mutant
mitochondrial N-proteomes led to the identification of 14 novel
substrates of Icp55, which were not identified in a previous study
(Vögtle et al., 2009).
The ChaFRADIC has also been employed to identify
MPP protein substrates in yeast S. cerevisiae mitochondria
(Burkhart et al., 2015). Using wild-type and mas1 mutant
(temperature-sensitive mutant of the MPP subunit Mas1)
mitochondria, 66 novel MPP substrates have been identified,
confirming the −2R position as a crucial determinant for MPP
recognition and processing (Burkhart et al., 2015).
Further, Carrie et al. (2015) employed ChaFRADIC in the
quantitative comparative N-terminome analysis of A. thaliana
mitochondria obtained from wild-type plants and mutants
lacking ICP55 or OCT1 mitochondrial protease. The group has
identified 88 mitochondrial proteins as putative ICP55 processing
substrates and characterized the ICP55 protease cleavage motif,
which appeared to be highly conserved between plants, yeast,
and mammals. In this study, seven putative substrates of OCT1
protease were also identified. However, they did not display
a consensus cleavage motif and did not reveal the presence
of the classical −10R motif characteristic for other eukaryotes
(Carrie et al., 2015).
Terminal Amine Isotope Labeling of
Substrates
Terminal Amine Isotope Labeling of Substrates was among the
first N-terminomics methods developed. Initially introduced by
the Overall lab (Kleifeld et al., 2010), TAILS has probably become
the most widely used in N-terminome analyses. In short, the
labeling of primary amines is typically performed through the
dimethylation of N-terminal α- and ε-amines. At this step,
stable isotope-labeled variants of formaldehyde can be introduced
to label primary amines (Demir et al., 2017). Stable isotope
labels can also be incorporated using amine-reactive isobaric
reagents such as iTRAQ or tandem mass tags (TMT) (Figure 3A).
Following trypsin digestion, the peptide mixture is incubated
with a high molecular-weight dendritic polyglycerol aldehyde
polymer (HPG-ALD) to separate N-terminal peptides from the
internal peptides (Figure 3B). In this reaction, internal peptides
covalently bind to the polymer, leaving the labeled N-terminal
peptides unbound. The polymer-bound internal peptides are
removed by centrifugation, while the N-terminal peptides are
further recovered by filtration and identified by LC-MS/MS (Luo
et al., 2019;Bogaert and Gevaert, 2020).
In the last years, TAILS has shown high robustness and
reliability proven by the independent application of this method
in numerous laboratories (Perrar et al., 2019). Furthermore,
moderately low amount of a starting material (0.1–1 mg of
protein per condition) and commercial availability of kits and
reagents are additional advantages of TAILS. Despite these certain
advantages, TAILS also has some limitations. For example, it
requires expensive patented aldehyde polymer and significant
statistical analyses to distinguish labeled and unlabeled N-termini
in the complex peptide mixture (Mintoo et al., 2021). Following
the principle of TAILS, the recent replacement of the HPG-
ALD polymer by N-hydroxysuccinimide beads in a novel iNrich
(integrated N-terminal peptide enrichment) workflow promises
higher accuracy and suitability for deep N-terminome profiling
(Ju et al., 2020).
The TAILS has been extensively used in N-terminome
profiling and protease substrate identification in many different
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Heidorn-Czarna et al. Mitochondrial Protein Processing
systems. In plants, TAILS has been employed to study the
N-terminal acetylation of cytosolic and plastid-located proteins
in the diatom Thalassiosira pseudonana (Huesgen et al., 2013)
and the N-terminome of A. thaliana chloroplasts (Rowland
et al., 2015). A similar approach has been taken for profiling
the cleavage site of transit peptides for protein import into the
cyanelles of the glaucophyte Cyanophora paradoxa (Köhler et al.,
2015a) and chloroplasts of A. thaliana (Köhler et al., 2015b).
Another TAILS-based study determined the impact of the Arg/N-
end rule pathway on the Arabidopsis root proteome and the seed
storage proteins (Zhang et al., 2015, 2018). So far, there is no
information about the use of TAILS in the N-terminome analysis
of plant mitochondria.
On the contrary, several TAILS-based studies have been
applied to investigate the N-terminome of human mitochondria.
For example, the modified version of TAILS termed MS-
TAILS (mitochondrial SILAC-TAILS) has been used to analyze
proteolysis in the mitochondria and parent cells during
initial apoptotic events before caspase-3 activation (Marshall
et al., 2018). This comprehensive approach has identified 206
mitochondrial proteins, which were not previously detected by
shotgun analyses. It also has revealed 475 unique mitochondrial
protein N-terminal peptides of all known mitochondrial
proteins, with 97 previously unknown proteolytic sites, which
constituted the highest reported coverage of the human
mitochondrial N-terminome. Quantitative comparative analysis
of mitochondrial and parental cell protein N-termini uncovered
altered levels of mitochondrial proteins implicated in protein
import, fission, and iron homeostasis, highlighting initial
proteolytic events in the mitochondrial pathway during apoptosis
(Marshall et al., 2018).
In another study, TAILS has been applied to profile
the mitochondrial N-terminome and identify candidate
mitochondrial protease activated by dopamine dysregulation in
neuroblastoma cells during the early stages of Parkinson’s disease
TABLE 3 | Mitochondrial processes regulated by limited proteolysis in yeast,
mammalian, and plant mitochondria.
Protease Mitochondrial functions
Yeast Mammals Plants
MPP Protein import and maturation
ICP55 Protein import and maturation
OCT1 Protein import and maturation
IMP Protein import and maturation Unknown
ATP23 OXPHOS functionality Unknown Unknown
Rhomboid Mitochondrial
fusion/fission,
morphology
Mitophagy, apoptosis Unknown
m-AAA Mitoribosome biogenesis
i-AAA Mitophagy Mitochondrial fusion/fission Unknown
OMA1 Unknown Mitochondrial fusion/fission,
integrated stress response
(IRS), apoptosis
Unknown
Details concerning the mitochondrial processing proteases and their protein
substrates are provided in Table 2.
(PD) (Lualdi et al., 2019). The authors have identified eleven
mitochondrial proteins with altered proteolytic processing,
and one of these proteins, the 39S ribosomal protein L38, was
cleaved by the neprilysin protease. In this study, for the first
time, neprilysin has been identified as a protease linked with
mitochondrial dysfunction upon the pathogenesis of Parkinson’s
disease, suggesting exploring targets of neprilysin as candidate
biomarkers of PD (Lualdi et al., 2019).
CONCLUSION AND PERSPECTIVES
The processing of mitochondrial proteins is a critical biological
mechanism in maintaining organellar and cellular homeostasis
by controlling protein location, abundance, and activity.
Mitochondria house diverse biochemical pathways controlled
by limited proteolysis, and many proteases are involved in
protein processing. While regulating constitutive mitochondrial
processes such as presequence cleavage and protein maturation
is highly conserved and relatively well understood, the
knowledge on condition-specific limited proteolysis is still
far from complete (Table 3). For example, the removal of
the mitochondrial targeting signals by MPP and IMP upon
protein import and additional protein processing by ICP55
and OCT1 is evolutionarily conserved throughout diverse
eukaryotes. The specific role of m-AAA in the biogenesis of
mitochondrial ribosomes also seems to be preserved across yeast,
mammals, and plants.
On the other hand, substrate processing by ATP23, rhomboid,
i-AAA, and OMA1 has rather diverse functions in yeast,
mammals, and plants, suggesting that molecular mechanisms of
limited proteolysis regulated by these mitochondrial proteases
evolved differently in distinct eukaryote lineages. In yeast,
the processing activity of rhomboid protease is required
in mitochondrial fusion, and the i-AAA-mediated limited
proteolysis is crucial in mitophagy. On the contrary, in
mammals, the processing activity of i-AAA has been linked
to mitochondrial fusion and fission, whereas the rhomboid
protease controls mitophagy and apoptosis. The regulation of
mammalian mitochondrial fusion and fission has also been
associated with the processing activity of OMA1. Recent
discoveries of the involvement of OMA1 in other processes,
such as apoptosis and the integrated stress response (IRS),
add new aspects to the understanding of the role of OMA1
as a critical processing protease in a variety of cellular
processes in mammals.
Our understanding of the mitochondrial limited proteolysis
in plants remains elusive despite numerous studies within the
last years that have expanded our knowledge on the role of
MPP, OCT1, ICP55, and m-AAA proteases in protein import and
maturation. To date, there is no experimental evidence on the
function of other mitochondrial proteases in limited proteolysis.
The characterization of protein N-termini generated from
the proteolytic cleavage is crucial in uncovering novel protein
substrates and understanding the physiological role of a
protease. However, despite recent progress in discovering new
processing peptidases and identifying protein processing as an
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Heidorn-Czarna et al. Mitochondrial Protein Processing
additional mode-of-action of general quality control proteases,
the substrate repertoire of such proteases is still relatively poor.
This work provided a brief overview of a few N-terminomics
techniques, COFRADIC, ChaFRADIC, and TAILS, which
dominated the N-terminomics field until recently and were
successfully employed to identify substrates of mitochondrial
processing proteases.
The field of N-terminomics has been rapidly expanding
over the last few years, promising more sensitive, faster,
and more accessible methods in the precise annotation
of the protease cleavage sites. Furthermore, the recent
development of targeted degradomics, which allows the accurate
quantification of cleaved products and an integrative approach
combining N- and C-terminomics techniques, will provide
a more comprehensive description of limited proteolysis
and the role of proteases in regulating mitochondrial and
cellular functions.
AUTHOR CONTRIBUTIONS
MH-C conceived and designed this study, prepared the
illustrations, and wrote the manuscript. AM assisted with
searching for literature and prepared the tables. HJ corrected the
final edition. All authors read and approved the final version
of the manuscript.
FUNDING
This work has been supported by a grant from the National
Science Centre, Poland (2017/27/B/NZ2/00558) and EPIC-XS,
project number 823839, funded by the Horizon 2020 Programe
of the European Union. Publication of this article was financially
supported by the Excellence Initiative – Research University
(IDUB) programme for the University of Wrocław.
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