Chloroplast immunophilins

Abstract

Immunophilins occur in almost all living organisms. They are ubiquitously expressed proteins including cyclophilins, FK506/rapamycin-binding proteins, and parvulins. Their functional significance in vascular plants is mostly related to plant developmental processes, signalling, and regulation of photosynthesis. Enzymatically active immunophilins catalyse isomerization of proline imidic peptide bonds and assist in rapid folding of nascent proline-containing polypeptides. They also participate in protein trafficking and assembly of supramolecular protein complexes. Complex immunophilins possess various additional functional domains associated with a multitude of molecular interactions. A considerable number of immunophilins act as auxiliary and/or regulatory proteins in highly specialized cellular compartments, such as lumen of thylakoids. In this review, we present a comprehensive overview of so far identified chloroplast immunophilins that assist in specific assembly/repair processes necessary for the maintenance of efficient photosynthetic energy conversion.

Full text

REVIEW ARTICLE
Chloroplast immunophilins
Ana TomašićPaić
1
&Hrvoje Fulgosi
1
Received: 2 February 2015 /Accepted: 30 April 2015
#Springer-Verlag Wien 2015
Abstract Immunophilins occur in almost all living organ-
isms. They are ubiquitously expressed proteins including
cyclophilins, FK506/rapamycin-binding proteins, and
parvulins. Their functional significance in vascular plants is
mostly related to plant developmental processes, signalling,
and regulation of photosynthesis. Enzymatically active
immunophilins catalyse isomerization of proline imidic pep-
tide bonds and assist in rapid folding of nascent proline-
containing polypeptides. They also participate in protein traf-
ficking and assembly of supramolecular protein complexes.
Complex immunophilins possess various additional function-
al domains associated with a multitude of molecular interac-
tions. A considerable number of immunophilins act as auxil-
iary and/or regulatory proteins in highly specialized cellular
compartments, such as lumen of thylakoids. In this review, we
present a comprehensive overview of so far identified chloro-
plast immunophilins that assist in specific assembly/repair
processes necessary for the maintenance of efficient photosyn-
thetic energy conversion.
Keywords Immunophilin .Photosystem II repair and
biogenesis .Thylakoid lumen .Auxiliary proteins .PPIases .
D1 protein
Introduction
Immunophilins are ubiquitous proteins found inall phyla from
prokaryotes to eukaryotes. They are involved in cellular reg-
ulatory pathways, signalling, protein folding, and protein traf-
ficking (Marks 1996;Heetal.2004;Romanoetal.2005).
Initially defined in 1989, when it was reported that previously
discovered cyclophilin and peptidyl-prolyl cis-trans isomer-
ase are identical proteins (Takahashi et al. 2003; Fischer et al.
1989), immunophilins comprise three functionally related
families: cyclophilins (CYP), FK506/rapamycin-binding pro-
teins (FKBP), and parvulins. Immunophilins bind immuno-
suppressive drugs cyclosporine A and FK506/rapamycin but
are not directly involved in immune responses in mammals.
While unrelated in their primary sequences, cyclophilins and
FKBPs possess peptidyl-prolyl cis-trans isomerase activity
(PPIase or rotamase activity) (Galat 2003), catalysing the
rate-limiting step of Xaa-Pro isomerization, from cis to trans
configuration of proline imidic peptide bonds. This step is
necessary for rapid protein folding, since rotation around pro-
line peptide bonds is energetically disfavoured due to their
partial double-bond character. Delocalization of amide nitro-
gen electrons results in approx. 22 kcal/mol energy barrier to
rotation and restrains the peptide bond in either cis or trans
configuration. Therefore, immunophilins are often described
as chaperone proteins that can even facilitate heat shock pro-
tein chaperone binding and release. Parvulins are not strictly
classified as immunophilins, since they do not bind immuno-
suppressants; however, they do exhibit the same rotamase
activity.
Immunophilins (PPIases, EC 5.2.1.8) can be divided into
either single-domain (contain only PPIase catalytic domain) or
multi-domain proteins. The molecular masses of single-
domain cyclophilins, FKBPs, and parvulins usually range be-
tween 18 and 21 kDa, 12 and 13 kDa, and 10 and 13 kDa,
Handling Editor: Jaideep Mathur
*Hrvoje Fulgosi
fulgosi@irb.hr
1
Division of Molecular Biology, Rudjer BoškovićInstitute, Bijenička
cesta 54, HR-10002 Zagreb, Croatia
Protoplasma
DOI 10.1007/s00709-015-0828-z

respectively. Complex immunophilins may harbour multiple
PPIase homology domains that can be active or inactive.
These PPIase homology domains are often found in three
tandem repeats. Further, complex immunophilins, especially
FKBPs, contain tetratricopeptide repeat (TPR) anti-parallel
helix-turn-helix domains that provide almost universal
docking surfaces for a multitude of protein partners, most
often the heat shock chaperone hsp90 (see below) (Aviezer-
Hagai et al. 2007). In addition, complex immunophilins pos-
sess additional functional regions such as leucine zippers,
beta-transducin repeat (WD40 repeat), ubiquitin ligase
activity-related protein domain or U-box domain, zinc finger
domain, RNA recognition motif,and Arg/Lysamino acid-rich
domain. Thus, complex immunophilins are frequent targets of
different molecular interactions.
In general, cyclophilins participate in a variety of cellular
processes and regulate functions such as apoptosis (Lin and
Lechleiter 2002), RNA processing (Krzywicka et al. 2001),
receptor signalling (Brazin et al. 2002), spliceosome and
RNA-induced posttranscriptional gene silencing complex as-
sembly (Smith et al. 2009), receptor complex stabilization
(Leverson and Ness 1998), control of mitosis by binding and
regulating of mitosis-specific phosphoproteins (Shen et al.
1998), detoxification of reactive oxygen species, and modula-
tion of calcium release activity, steroid receptor activity, de-
velopmental regulation (Patterson et al. 2000), and pathogen-
host interactions (Deng et al. 1998), as previously reviewed
(Kumari et al. 2013). Mammalian FKBPs associate through
TPR domains with hsp90, known as an abundant chaperone
that interacts with a broad array ofprotein clients and regulates
numerous essential cellular pathways (Cox and Smith 2007).
However, mammalian parvulins were found to participate in
pre-RNA maturation (Lavoie et al. 2001), in cancer formation
(Lu et al. 2006), and in progression of chronic diseases, such
as Alzheimer’s and Parkinson’s disease (Butterfield et al.
2006;Driveretal.2014).
Immunophilins in plants
Plants possess the largest immunophilin families found in eu-
karyotes, with single- and multi-domain members varying in
functions and cellular localization. The first vascular plant
cyclophilin sequences were identified in the early 1990s, from
tomato (Solanum esculentum), maize (Zea mays), and oilseed
rape (Brassica napus) (Gasser et al. 1990). Plant
immunophilins are characterized by their conserved PPIase
domains and are, akin to mammalian isoforms, implicated in
protein folding, assembly, and protein trafficking. The se-
quencing of the Arabidopsis genome, associated with the
elaborate proteomic analyses, allowed the identification of
genes encoding numerous putative immunophilin isoforms.
Hitherto, Arabidopsis genome encodes 58 putative genes for
immunophilins: 35 cyclophilins (Trivedi et al. 2012), 23
FK506-binding proteins, and 3 genes encoding parvulins
(Rahfeld et al. 1994). Arabidopsis putative cyclophilin iso-
forms have sequences that are predicted to be localized in
almost all cellular compartments: 14 in the cytosol, 9 in chlo-
roplasts, 2 in the endoplasmic reticulum, 2 in mitochondria, 3
in the nucleus, 1 in plasmodesmata, 1 in the Golgi apparatus,
and 3 in the plasma membrane (Trivedi et al. 2012).
Arabidopsis cyclophilins can interact with the bacterium vir-
ulence proteins (VirD2 and VirE2), essential for the precise
integration of T-DNA complexes into host plant cells (Deng
et al. 1998). Rice (Oryza sativa ssp. Japonica) and soybean
(Glycine max) are the two additional plant species having the
highest number of putative cyclophilins identified: 28 in rice
(Trivedi et al. 2012; Ahn et al. 2010) and 62 in soybean, as
reviewed previously (Mainali et al. 2014).
Detailed characterization of the FK506-binding proteins,
principally the most extensively studied class of plant
PPIases, identified 23 putative FKBP isoforms in
Arabidopsis,16single-domainAtFKBP and 7 multi-domain
isoforms, and their additional domain homologues. In the
scope of these studies, their probable multifunctional proper-
ties have also been proposed. Additional 29 FKBP polypep-
tides were found in rice. FK506-binding proteins have not yet
been identified in legume species; however, they may soon be
revealed in various genome-wide projects. Plant multi-domain
FKBPs can often contain already-mentioned TPR domains,
proline-rich or proline-containing motifs (designated as WW
domains), and/or calmodulin-binding motifs in addition to the
obligatory FK506-binding domain. Briefly, the TPR motif is a
34-amino acid consensus sequence commonly occurring in
multiple copies within the same protein. Proteins with this
motif are involved in a variety of cellular functions
(D’Andrea and Regan 2003). Mammalian FKBPs frequently
associate via TPRs with the hsp90. In plants, TPR-mediated
interactions have been implicated in hormone responses (gib-
berellin, cytokinin, and auxin) as well as the biosynthesis of
ethylene, as previously reviewed (Schapire et al. 2006).
Several genes encoding TPR proteins may have roles in biotic
defence responses (Takahashi et al. 2003), photosystem I as-
sembly (Stockel et al. 2006), and messenger RNA (mRNA)
processing and stability (Boudreau et al. 2000; Vaistij et al.
2000).
Multi-domain FKBPs occur in the cytosol (AtFKBP42,
AtFKBP62, AtFKBP65) and nucleus (AtFKBP43,
AtFKBP53, AtFKBP72) of Arabidopsis. Nuclear isoforms
have either unknown functions (AtFKBP43) or may function
by interacting with hsp90 (AtFKBP72, designated also as
DEI1, PAS1, PASTICCINO1) (He et al. 2004). A new class
of developmental mutants affected in cytokinin response
known as the pasticcino (pas) mutants has several develop-
mental abnormalities at the embryonic stage (thick hypo-
cotyls, bushy rosettes, infertile flowers) even in the absence
of cytokinins (Vittorioso et al. 1998). FKBP-like pas1 genes
A.T. Paić,H.Fulgosi

are required for coordination of cell division and proliferation
during plant development (Harrar et al. 2001), two processes
tightly regulated by cytokinins and auxins. Recently charac-
terized AtFKBP53 is a chromatin-modulating factor required
for suppression of ribosomal RNA gene expression (Li and
Luan 2010).
Multi-domain AtFKBP42 protein found in cytosol and pre-
dicted to be localized in plasma membrane is characterized as
an inactive PPIase (Kamphausen et al. 2002). Through its
specific domains, AtFKBP42 interacts with the plasma
membrane-localized ATP-binding cassette (ABC-B/multi-
drug resistance/P-glycoprotein superfamily) transporters,
most likely involved in auxin transport (Cho and Cho 2013).
Mutants with disrupted AtFKBP42 alleles, FKBP-like twisted
dwarf 1 (TWD1) (Bouchard et al. 2006) and ultracurvata 2
(UCU2) (Pérez-Pérez et al. 2001), are phenotypically indistin-
guishable and have a dwarf phenotype, twisted appearance,
and reduced fertility (Kamphausen et al. 2002; Geisler et al.
2003). Biochemical analyses of AtFKBP42 twisted dwarf 1
(twd1)-null mutants suggested that TWD1 has a role in
brassinosteroid reception or signal transduction (Geisler
et al. 2003).
The multi-domain isoforms of AtFKBP62 and AtFKBP65,
known as ROF1 and ROF2, which stands for Brotamase
FKBP^, occur as homologous duplicates in plant genomes
(Vucich and Gasser 1996). Proteins ROF1 and ROF2 are in-
volved in response to heat stress through their interaction with
hsp90 in Arabidopsis and the heat shock transcription factor
HsfA2 (Meiri et al. 2010), as reviewed in Gollan et al. (2012).
The FKBP protein ROF1 (AtFKBP62) is differentially regu-
lated and developmentally controlled in response to heat stress
and binds the hsp90 via TPR domain (Aviezer-Hagai et al.
2007). This protein shares 34 % sequence identity with the
amino acid sequence of ultracurvata (UCU2) Arabidopsis mu-
tant, already known to be involved in essential auxin and
brassinosteroid signalling (Pérez-Pérez et al. 2004).
Moreover, ROF1 immunophilin exhibits chaperone activity
and regulates steroid hormone signalling in plants. This robust
system orchestrates genome expression, signal transduction,
metabolism, defence, and development, all molecular mecha-
nisms crucial for plant growth regulation.
Plant parvulin-type PPIases have been identified from
Arabidopsis;PIN1At (Landrieu et al. 2000), apple (Malus
domestica); MdPin1 (Yao et al. 2001), woolly foxglove
(Digitalis lanata); and DlPar13 (Metzner et al. 2001), the
model legume Lotus japonicus; annotated LjPar1, LjPar2,
and LjPar3 (Kouri et al. 2009). All Lotus parvulins (Kouri
et al. 2009)andArabidopsis PIN1 (Wang et al. 2010)were
biochemically and/or genetically characterized including their
functions proposed. Other parvulin biological functions are
still unknown. Parvulins can be further divided into the
PIN1-type (phosphorylation-dependent) and the non-PIN1-
type (phosphorylation-independent) parvulins according to
their substrate specificity (Lu et al. 2007). The Arabidopsis
genome contains three parvulin genes (He et al. 2004): a
single-domain PIN1At (At2g18040) and multi-domain
PIN2At (At1g26550) and PIN3At (At5g19370). The PIN1-
type parvulin in Arabidopsis,PIN1At, contains a single
PPIase domain but lacks the proline-containing motifs (WW
domain) at the N-terminus. PIN1At is involved in the isomer-
ization of key transcription factors important for flowering
regulation (Wang et al. 2010). PIN3At also harbours a C-
terminal rhodanese-like domain, as well as predicted N-
terminal targeting sequence (He et al. 2004). Experiments in
a cell-free system indicate that, similarly to FKBPs,
parvulin(s) in Arabidopsis is/are probably implicated in the
inhibition of auxin signalling (Dharmasiri et al. 2003).
To conclude, although extensively studied in recent years,
little is known regarding the exact roles of immunophilins in
plants. Particularly intriguing are findings of PPIase activity in
highly specialized cellular compartments, such as the thyla-
koid lumen of chloroplasts. In this review, we particularly
focus on chloroplast-localized immunophilins and their func-
tions, especially those that are involved in photosynthetic en-
ergy conversion processes.
Plastid immunophilins
Immunophilins of chloroplast stroma
The only presently recognized nuclear-encoded, single-
domain cyclophilin targeted to stroma of Arabidopsis chloro-
plasts is AtCYP20-3 (ROC4 or rotamase CYP4) (Lippuner
et al. 1994). It is active under reducing conditions, and its
synthesis is strongly induced by light (Lippuner et al. 1994).
This 28.2-kDa polypeptide has been implicated in protein
folding and assembly in response to light and redox changes
(Dominguez-Solis et al. 2008).Conservedcysteineresidues
have a proposed function in redox regulation forming two
disulphide bonds under oxidizing conditions, Cys
54
–Cys
171
and Cys
129
–Cys
176
(Motohashi et al. 2003), and are important
for the isomerization of the ROC4 (Motohashi et al. 2003).
Cys
129
and Cys
171
mayalsointeractwithdifferent
chloroplast-located peroxiredoxins (PrxA and PrxB) (Laxa
et al. 2007). Lincomycin experiments (inhibitor of protein
synthesis in chloroplasts) on roc4-knock-out plants suggest
that ROC4 functions in repair of photodamaged PSII (Cai
et al. 2008). roc4 plants are sensitive to high-light stress,
which may be explained by the inhibition of the PSII repair
cycle. Furthermore, ROC4 seems to link photosynthetic elec-
tron transport chain and thylakoid redox status with the fold-
ing of chloroplast serine acetyltransferase (SAT1), a rate-
limiting enzyme in cysteine biosynthesis (Dominguez-Solis
et al. 2008).
Chloroplast immunophilins

Three recently described chloroplast cyclophilins (Trivedi
et al. 2012), with their locus numbers: At2g16600 (AtCYP19-
1, cyclophilin 19, Cyp19, ROC3), At4g34870 (AtCYP1,
ROC5), and At5g58710 (ROC7), based on biological data-
base mining research have been localized and classified.
Although their domains, function, and subcellular localization
data are a matter of future research, in the work of Stangeland
et al. (2005), AtCYP19-1 chloroplast stroma cyclophilin, or
ROC3, was suggested to have a role in seed development.
Chloroplast-targeted single-domain FKBPs have not been
identified so far in the Arabidopsis genome. However, a dis-
tantly related multi-domain member of the FKBPs, designated
trigger factor,AtTIG, was found by the Arabidopsis genome
sequencing project. AtTIG (locus At5g55220) is predicted to
be localized in chloroplast stroma. Its function has been pro-
posed solely based on its similarity to bacterial homologues.
In bacteria, trigger factor is defined as a ribosome-associated
chaperone that might function in protein synthesis and
targeting processes (Hesterkamp and Bukau 1996).
Previously mentioned rhodanese domain-containing
parvulin, PIN3At, appears to be targeted to chloroplast stroma
(He et al. 2004).
Lumen-located single-domain cyclophilins and FKBPs
The dual genetic origin and the enormous physiological ver-
satility, in particular its ability to manage short- and long-term
changes in light environment, are outstanding characteristics
of photosynthetic membranes. A striking feature of the
immunophilins found in chloroplasts is their particular abun-
dance in the thylakoid lumen, rather than in the stroma or in
the intermembrane space of the envelope. Sixteen
immunophilin members can be found in the lumen of
Arabidopsis, but only two, AtCYP20-2 and AtFKBP13, pos-
sess PPIase activity. Interestingly, AtFKBP13 sustains all
PPIase activities in the thylakoid lumen when AtCYP20-2 is
inactivated in Arabidopsis (Edvardsson et al. 2007), while
both enzymes are not essential for plant development
(Ingelsson et al. 2009). These findings brought about the
new concept for the functions of lumenal immunophilins, pos-
tulating that their functional roles are not related to their en-
zymatic activity but are associated with other regulatory and
signalling mechanisms and cellular functions (Ingelsson et al.
2009). The lumenal compartment is the place where photo-
synthetic water splitting, oxygen evolution, and redox chem-
istry converge. In this context, it has been recognized that
AtFKBP13 contains a pair of disulphide bonds that are essen-
tial for its PPIase activity (Gopalan et al. 2004). These redox-
active bonds are unique to chloroplast FKBPs and are absent
in animal and yeast homologues. Further, they can be reduced
by thioredoxin, rendering AtFKBP13 enzymatically inactive
(Gopalan et al. 2004). Thus, a previously unrecognized para-
digm for redox regulation in which activation by light is
accomplished in synchrony with oxygen evolution by the ox-
idation of sulfhydryl groups (SH to S–S conversion) has been
proposed (Gopalan et al. 2004). This mechanism specific for
the thylakoid lumen is in direct contrast to regulation of en-
zymes in the stroma, where activation and light-regulated in-
crease in activity of various enzymes and metabolic pathways
are triggered by reduction of disulphides via thioredoxin (S–S
to SH conversion).
The four single-domain cyclophilin isoforms, AtCYP20-2,
AtCYP26-2, AtCYP28, and AtCYP37, occur in Arabidopsis
chloroplast lumen and contain N-terminal bipartite (chloro-
plast/thylakoid lumen) targeting sequences. The AtCYP37
and the multi-domain AtCYP38 have highly diverged consen-
sus sequences, but the most unorthodox are the AtCYP26-2
and the AtCYP28, containing only 1 to 3 out of 13 conserved
residues crucial for PPIase activity and inhibitor binding.
Protein sequencing, import assays, and immuno-detection
techniques have experimentally verified the predicted chloro-
plast localization of all four of these polypeptides. The
AtCYP26-2 and the AtCYP28 possess a highly conserved
twin-arginine leader motif (S/TRRXFLK) within the N-
terminal signal peptide of these proteins. Fully folded proteins
with the twin-arginine motif are targeted to the lumenal com-
partment of thylakoid membranes via the twin-arginine-
dependent protein translocation pathway or briefly Tat
(Schnell 1998). Formerly termed the ΔpH-dependent path-
way, Tat is essential for photosynthesis and is required for
the assembly of PSII and the cytochrome b
6
f complexes
(Cyt b
6
f) (Palmer and Berks 2012).
The AtCYP20-2 gene expression is light regulated, while
the protein itself has PPIase activity mainly in the lumen
(Edvardsson et al. 2003). Initially, AtCYP20-2 was associated
with PSII (Romano et al. 2004); however, functional analyses
of PSII in cyp20-2 mutant plants did not reveal any problems
with PSII, neither under growth-light nor high-light condition
(Sirpiö et al. 2009). Instead, it has been demonstrated that
AtCYP20-2 associates with thylakoid membrane-embedded
NAD(P)H dehydrogenase (NDH) complexes (Sirpiö et al.
2009). NDH complexes are regulated via redox signals and
mediate cyclic electron transport in photosynthesis, as well as
chlororespiration. The accumulation of AtCYP20-2 was stud-
ied both in wild-type (WT) and in several mutants affecting
various components of the NDH complex, specifically, ndho
mutants lacking the hydrophilic NDHO protein, mutants with
defects in CRR2 nucleus-encoded factor important for proper
expression of the hydrophobic NDHB subunit, and ndh45
lacking the NDH45 component embedded into the hydropho-
bic domain of the NDH complex. The accumulation of
AtCYP20-2 was strongly reduced in NDH mutant plants with
affecting hydrophobic domain of the supercomplex (crr2-2
and ndh45). AtCYP20-2 probably functions as an auxiliary
protein in the biogenesis of NDH complexes and is therefore
considered to be involved in both cyclic electron transport and
A.T. Paić,H.Fulgosi

chlororespiration (Sirpiö et al. 2009). Such involvement of
lumen cyclophilin is unexpected since chlororespiration en-
zymes sit at the stromal face of the membrane, which is also
the side where reducing equivalents are reinjected in cyclic
electron flow. However, the assumption is supported by the
finding that F
0
rise of chlorophyll afluorescence, generated
by NDH-mediated reduction of the plastoquinone pool in
darkness, were somewhat decreased in the cyp20-2 mutants
as compared to WT (Sirpiö et al. 2009).
The other two complex cyclophilins occur in the lumen,
single-domain AtCYP37 (protein of an unknown function)
and complex AtCYP38 (discussed in detail below). They were
identified using genomics and proteomics (Peltier et al. 2002).
There are 11 additional single-domain FK506-binding pro-
teins (AtFKBP13, AtFKBP16-1, AtFKBP16-2, AtFKBP16-3,
AtFKBP16-4, AtFKBP17-1, AtFKBP17-2, AtFKBP17-3,
AtFKBP18, AtFKBP19, AtFKBP20-2) with predicted lumenal
localization (He et al. 2004;Romanoetal.2005; and refer-
ences therein). Interestingly, in the thylakoid lumen, only
AtFKBP13 and AtCYP20-2 are active PPIases (Edvardsson
et al. 2007; Shapiguzov et al. 2006; Ingelsson et al. 2009).
The best studied is AtFKBP13 (synonyms: FKBP22-1,
FKBPK), which is considered as a protein folding catalyst.
AtFKBP13 translocates to the lumen via the already-
mentioned Tat pathway independently of its redox status.
AtFKBP13 is involved in the integration of the Rieske iron–
sulphur protein into the Cyt b
6
f complex (Gupta et al. 2002).
This insertion appears to be a rate-limiting step in the assem-
bly, most likely influencing the upstream trafficking of Rieske
preprotein. Also, AtFKBP13 might contribute to Fe–Scluster
formation in the Rieske protein (Edvardsson et al. 2007). In
AtFKBP13 mRNA interference plants, Rieske precursors ac-
cumulate in the cytosol or chloroplast stroma (Gupta et al.
2002). Both yeast two-hybrid and in vitro protein–protein in-
teraction assays suggested the interaction between Rieske and
the pre-AtFKBP13,aswellastheAtFKBP13 import interme-
diate containing only thylakoid-targeting presequence, but not
with the mature protein (Gupta et al. 2002). In contrast, only
the interaction of Rieske and the mature AtFKBP13 was de-
tected in situ, implying their interaction within the lumen
(Gollan et al. 2011). The crystal structure of AtFKBP13 at
1.88-Å resolution revealed a pair of conserved disulphide
bonds, present at the N- and C-termini of the protein. These
might be involved in redox regulation by thioredoxin
(Gopalan et al. 2004; Gopalan et al. 2006). These disulphide
bonds seem also to be essential for protein’sPPIaseactivity.
AtFKBP16-2 is an inactive PPIase not directly involved in
redox-dependent reactionsin the lumen, despite having a high
sequence similarity to AtFKBP13. Akin to AtCYP20-2,
AtFKBP16-2 has recently been found to form interactions
with the lumen-exposed region of the thylakoid NDH com-
plex (Peng et al. 2009). Acting as an additional NDH com-
plex, AtFKBP16-2 may also have a role in the maintenance of
cyclic electron transport (Peng et al. 2009). Recently,
AtFKBP16-2 has been designated as a member of cargo pro-
teins containing ILV motif (Khan et al. 2013). Cargo proteins
are known to associate with vesicle transport system and pos-
sess specialized signals for recognition and interaction with
receptors and membranes (Khan et al. 2013). Further, an
AtFKBP16-1 ortholog in wheat (Triticum aestivum),
TaFKBP16-1, participates in early chloroplast development
(Gollan and Bhave 2010) and may mediate response to pho-
tosynthetic stress, by regulating the stability of PSI component
PsaL in Arabidopsis (Seok et al. 2014). Other two AtFKBP16
isoforms found in chloroplast lumen await further
characterization.
The three putative FKBP-type peptidyl-prolyl isomerases,
AtFKBP17-1, AtFKBP17-2, and AtFKBP17-3, are also local-
ized in the thylakoid lumen. All three proteins have no detect-
able PPIase activity and have so far no determined functions.
Their orthologs were sequence identified in rice (OsFKBP17-
1, OsFKBP17-2) and maize (Z. mays;ZmFKBP17-1,
ZmFKBP17-2a, ZmFKBP17-2b) (Wang et al. 2012). Further
lumenal FKBPs with yet unassigned functions, AtFKBP18
and AtFKBP19, contain only one or no cysteine residues
and thus are not likely to be redox regulated.
Lumen-located complex cyclophilins
Although the 40-kDa thylakoid lumen PPIase TLP40 (Fulgosi
et al. 1998; Vener et al. 2000) was reported as the first complex
immunophilin found in vascular plant chloroplasts (Fulgosi
et al. 1998), already in 1992, the group of A. Mattoo has
reported that plant organelles contain distinct peptidyl-prolyl
cis-trans isomerases (Breiman et al. 1992). Isolated from spin-
ach, TLP40 contains a loosely conserved C-terminal
cyclophilin domain, two predicted centrally positioned leu-
cine zippers, and tandemly arranged phosphatase-binding se-
quences. It has been proposed that the phosphatase-binding
stretches are important for TLP40 interaction with the putative
PP2A-like phosphatase (Vener et al. 2000; Rokka et al. 2000)
involved in dephosphorylation of the PSII core proteins.
While TLP40 is an active PPIase (Fulgosi et al. 1998), its
Arabidopsis homologue AtCYP38 lacks PPIase activity
(Edvardsson et al. 2003; Shapiguzov et al. 2006), probably
due to the substituted amino acid residues in the conserved
catalytic domain. These amino acid exchanges might affect its
substrate binding properties. However, akin to the TLP40,
putative phosphatase-interacting regions have been predicted
at the N-terminus of AtCYP38, leading to the conclusion that
AtCYP38 also acts as a regulator of a thylakoid-associated
phosphatase. This PP2A-like phosphatase is postulated to de-
phosphorylate both CP43 antenna and PSII core proteins.
Lateral protein distribution studies indicate that AtCYP38 ac-
cumulates near the lumen-exposed moiety of the D1 protein,
where AtCYP38 probably assists in the rapid turnover of the
Chloroplast immunophilins

D1 (Sirpiö et al. 2008;Fuetal.2007). Furthermore, AtCYP38
is involved in a rather intricate assembly of oxygen-evolving
complex (Sirpiö et al. 2008)(Fig.1a, b), where it assists the
proper folding of nascent D1 protein in PSII, simultaneously
affecting the correct assembly of water-splitting Mn
4
–Ca clus-
ters (Sirpiö et al. 2008). The AtCYP38 inactivation (gene
At3g01480) influences the dephosphorylation of PSII, partic-
ularly affecting folding properties of the D1, and probably also
of the CP43. Analyses of the AtCYP38 crystallographic data
at the 2.39-Å resolution revealed an additional and perhaps
intricate interaction with the E-loop of PSII chlorophyll pro-
tein 47 (CP47) (Vasudevan et al. 2012).
The AtCYP38-knock-out plants grown under light–dark
regime exhibit dramatic retardation of growth, small leaves,
and poor seedling survival (Sirpiö et al. 2008;Fuetal.2007;
Lepedušet al. 2009). The observed stunned phenotype is
however abolished in conditions of continuous illumination.
The AtCYP38 knock-outs were found highly susceptible to
photodamage, accompanied with a reduced PSII activity and
impaired fine-tuning of the oxygen-evolving complexes
(Sirpiö et al. 2008).
Multi-domain FKBPs seem not to be present in the
Arabidopsis chloroplast lumen.
AtCYP38 in PSII repair and assembly mechanisms
Although the whole repair process for PSII is far beyond the
role of immunophilins and the scope of this review, for the
purpose of comprehensiveness, here, we summarize the most
important events in the PSII repair cycle. As depicted in Fig. 1,
a
b
Fig. 1 Schematic representation of the AtCYP38 and associated
auxiliary enzyme functional roles in the main steps of PSII repair and
reassembly under high-light illumination. The structures of the PSII
complexes and related polypeptides within the core of the complex are
depicted (CP43, CP47, D1, and D2). a,bThemainstagesofdenovo
assembly and repair of PSII in chloroplasts, including the process of
degradation of photodamaged PSII protein subunits, mainly the D1
subunit, its de novo protein synthesis, membrane reinsertion and
folding of the nascent protein, and final reassembly of the released
proteins into the reconstituted PSII. Many of the auxiliary and
regulatory proteins required for sequential repair steps represented here
are the following: AtCYP38, cyclophilin protein 38; HSP70, heat shock
protein 70; HCF136, PSII assembly factor (high chlorophyll fluorescence
136); LPA1, chloroplast membrane chaperone; ELIPs, early light-
inducible proteins; LPA19, low PSII accumulation protein; Psb27
assembly factor including chloroplast proteases: CtpA, carboxyl-
terminal processing protease; Deg, serine-type protease; FtsH, ATP-
dependent metalloprotease complex; and thylakoid lumen phosphatase
AtTLP18.3. See text for further details. Redox-controlled thylakoid
protein phosphorylation as well as nucleotide-dependent reactions in the
chloroplast thylakoid lumen, including nucleoside triphosphate
transporters between the lumen and stroma (a thylakoid ATP/ADP
carrier (TAAC)), nucleoside diphosphate kinase 3 (NDPK3), and a
thylakoid phosphate transporter (PHT4;1) are all integrated in the scheme
A.T. Paić,H.Fulgosi

the PSII dynamically regulated repair cycle in chloroplasts is
initiated due to the excess light-induced photoinactivation of
D1 PSII reaction centre proteins (Fig. 1a), followed by the
related degradation, partial disassembly of damaged PSII
monomers, and their migration to stroma-exposed thylakoids
(Fig. 1b). In stroma thylakoids, the cooperative D1 degrada-
tion by the specific proteases (FtsH and Deg) is assisted by
membrane-anchored thylakoid lumen-exposed protein
AtTLP18.3 that possesses in vitro serine/threonine phospha-
tase activity (Sirpiö et al. 2007;Wuetal.2011). Thylakoid
proteins CP47, PsbP, PsbQ, PsbR, and PsaF are phosphory-
lated on serine or threonine residues, and this phosphorylation
appears to take place on the lumenal side of the photosynthetic
membranes (Pesaresi et al. 2011). The evidence of AtTLP18.3
phosphatase activity in vivo is hitherto not available; there-
fore, the significance of this type of posttranslational modifi-
cation in the lumen should be interpreted with caution.
Upon high-intensity illumination, state transition protein
kinase 8 (STN8 PK), which is located on the stroma-
exposed membranes, becomes activated and at least partially
involved in phosphorylation of PSII core subunits (D1, D2,
CP43, PsbH). These proteins are subsequently dephosphory-
lated by the PP2C-type PSII core phosphatase (PBCP) (not
shown). PSII core phosphatase efficiently dephosphorylates
several core subunits of PSII (Fig. 1b) (Samol et al. 2012;
Puthiyaveetil et al. 2014). Importantly, dephosphorylation of
PSII core proteins is mediated by specific intrinsic and extrin-
sic phosphatases and is independent of the LHCII phosphatase
(Vener et al. 2000; Rokka et al. 2000).
AtCYP38 acts as an auxiliary component and assists the
assembly of PSII core monomers during biogenesis and repair
(Sirpiö et al. 2008;Fuetal.2007). Additionally, AtCYP38
interacts with the PSII apoprotein CP47, participating in its
proper folding and integration into PSII (Vasudevan et al.
2012). AtCYP38 also functions as a negative regulator of the
intrinsic thylakoid protein phosphatase essential for PSII core
subunit dephosphorylation (Vener et al. 2000; Rokka et al.
2000). AtCYP38 is released into the lumen upon PP2A-like
protein phosphatase activation (Fig. 1b, step 2). It is then
attached back to PSII subunits (step 3) where it assists in
efficient degradation of D1, its de novo synthesis (step 3),
and final reassembly into the PSII electron donor side (step
4) (Sirpiö et al. 2008;Muloetal.2008,2012). Several further
auxiliary enzymes are involved as well: LPA1 functions as an
integral membrane chaperone (Peng et al. 2006), HCF136 is
an assembly factor (Plücken et al. 2002), and chaperone
HSP70 assists in psbA mRNA translation. All are involved
in coordination of D1 protein folding, which makes them es-
sential for PSII assembly. The CtpA protease, with the aid of
LPA19 protein homologue of PSB27, facilitates carboxyl-
terminal processing of the D1 protein during PSII biogenesis,
resulting in proper assembly of a manganese cluster
(Oelmüller et al. 1996; Wei et al. 2010). The early light-
inducible proteins (ELIPs) might serve as a temporary storage
of free pigments during the PSII reassembly (Valledor et al.
2012). Similarly to AtCYP38, assembly factor PSB27 is also
implicated in the biogenesis and repair of light-damaged PSII,
in a way of assisting the correct assembly of the oxygen-
evolving complex into a multi-subunit PSII supercomplex
(step 4). After the PSII core monomers are reassembled to-
gether with the structural and peripheral antenna complexes in
grana thylakoids, the oxygen-evolving complex is activated
(Fig. 1a) and iron–sulphur clusters of the cytochrome f (Cyt f)
and plastocyanin (PC) proteins are reoxidized. The activation/
deactivation of the STN8 PK is regulated by redox-induced
structural changes of Cyt b
6
f complex caused by plastoqui-
none binding to the quinol oxidation site (Q
o
)(Veneretal.
1998;Zitoetal.1999). Further, PSII dimerization and accu-
mulation of PSII supercomplexes are assisted by the
AtFKBP20-2 and AtPSB29 proteins (not shown) (Lima et al.
2006). Red and ox indicate reduced or oxidized forms of elec-
tron carriers.
As indicated in Fig. 1b, chlorophyll-binding protein CP47
is phosphorylated in the lumen-exposed loop by still unknown
kinase(s) (Pesaresi et al. 2011). The physiological role of this
ATP-dependent phosphorylation is not yet understood. These
unidentified kinase(s) are also capable of phosphorylating dis-
sociated components of the oxygen-evolving complex (PsbQ,
PsbR, PsbP), except an extrinsic subunit PsbO (O) (Spetea
and Lundin 2012).
Other implications and conclusions
Our understanding of immunophilin molecular functions in
plants has rapidly increased, thanks to the development of
novel genetic, genomic, and proteomic approaches. Plant
immunophilins are influenced by a variety of biotic or abiotic
factors and are involved in an array of molecular processes,
ranging from cell signalling to plant development.
Cyclophilins and FKBPs are rather numerous in the lumenal
compartment of vascular plant chloroplasts, where they are
mostly involved in repair and assembly of photosynthetic su-
pramolecular complexes, thereby assisting in chloroplast bio-
genesis and in maintenance of efficient energy conversion.
Despite being extensively studied, little is known about the
exact molecular targets of these specialized auxiliary proteins.
The regulatory mechanisms governing the activity of complex
lumenal immunophilins are equally ambiguous. The future
challenges in chloroplast immunophilin research include iden-
tification of immunophilin-containing regulatory
supercomplexes, mechanisms of their posttranslational modi-
fications, reasons and consequences of possible complex
immunophilin oligomerization, effects of immunophilin over-
expression in model plants, and elucidation of the so far
Chloroplast immunophilins

undetermined functions of cyclophilins and FKBPs found in
the stroma and in the inner membrane spaces.
Acknowledgments The work in the HF laboratory is supported by the
grant CRP/CRO11-01 from the International Centre for Genetic Engi-
neering and Biotechnology (ICGEB). We thank Professor Jonathan
Gressel, Weizmann Institute of Science, Israel, for English language
editing and critical reading of the manuscript. We thank Lucija Horvat
for further language improvements of the manuscript.
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