Available via license: CC BY-NC-SA 4.0
Content may be subject to copyright.
Available via license: CC BY-NC-SA 4.0
Content may be subject to copyright.
The Journal of Cell Biology
The Rockefeller University Press, 0021-9525/2003/01/201/12 $8.00
The Journal of Cell Biology, Volume 160, Number 2, January 20, 2003 201–212
http://www.jcb.org/cgi/doi/10.1083/jcb.200207045
JCB
Article
201
The coiled-coil membrane protein golgin-84 is a novel
rab effector required for Golgi ribbon formation
Aipo Diao,
1
Dinah Rahman,
3
Darryl J.C. Pappin,
3
John Lucocq,
2
and Martin Lowe
1
1
School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK
2
School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
3
Proteomics Section, Medical Research Council Clinical Sciences Centre, Imperial College of Science, Technology, and Medicine,
Hammersmith Hospital, London W12 0NN, UK
ragmentation of the mammalian Golgi apparatus during
mitosis requires the phosphorylation of a specific subset
of Golgi-associated proteins. We have used a biochem-
ical approach to characterize these proteins and report
here the identification of golgin-84 as a novel mitotic target.
Using cryoelectron microscopy we could localize golgin-84
to the cis-Golgi network and found that it is enriched on
tubules emanating from the lateral edges of, and often
connecting, Golgi stacks. Golgin-84 binds to active rab1
F
but not cis-Golgi matrix proteins. Overexpression or depletion
of golgin-84 results in fragmentation of the Golgi ribbon.
Strikingly, the Golgi ribbon is converted into mini-stacks
constituting only
25% of the volume of a normal Golgi
apparatus upon golgin-84 depletion. These mini-stacks are
able to carry out protein transport, though with reduced
efficiency compared with a normal Golgi apparatus. Our
results suggest that golgin-84 plays a key role in the assembly
and maintenance of the Golgi ribbon in mammalian cells.
Introduction
The Golgi apparatus of mammalian cells consists of stacked
cisternae that are connected laterally by tubules to form a
continuous ribbon (Rambourg and Clermont, 1997). Each
face of the Golgi stack is flanked by tubulo-reticular net-
works that act as entry (cis-Golgi) or exit (trans-Golgi) sorting
stations. Proteins that enter the cis-Golgi network are either
recycled back to the ER or transported onwards through the
Golgi stack where they can be modified by the sequential
action of enzymes present in each of the individual cisternae.
Upon arrival at the trans-Golgi network, proteins are pack-
aged into carriers for delivery to the plasma membrane or
endosomes. How the Golgi apparatus retains its identity and
compartmental organization in spite of the extensive mem-
brane and protein flux through this organelle is not clear,
but at least two classes of protein complexes appear to be
involved: the Golgi spectrin/ankyrin skeleton (De Matteis
and Morrow, 2000) and the Golgi matrix proteins (Seemann
et al., 2000a; Pfeffer, 2001).
The Golgi matrix was originally identified as a detergent-
insoluble structure with the ability to bind Golgi enzymes
(Slusarewicz et al., 1994). Several components of the Golgi
matrix have now been identified. The best characterized are
p115, the GM130–GRASP65 complex, and the integral
membrane protein giantin (Linstedt and Hauri, 1993;
Nakamura et al., 1995; Sapperstein et al., 1995; Barr et al.,
1997). GRASP65 is a cisternal stacking protein that also acts
as a receptor for the coiled-coil protein GM130, targeting it
to the cis-Golgi (Barr et al., 1997, 1998). GM130, in turn,
is a receptor for p115, a coiled-coil protein that is required
for the tethering of transport vesicles to Golgi cisternae
(Nakamura et al., 1997; Nelson et al., 1998). Giantin also
binds to p115 and participates in vesicle tethering, and,
interestingly, both p115 and giantin together with GM130
are required for stacking of Golgi cisternae in vitro (Sönnichsen
et al., 1998; Shorter and Warren, 1999). The interactions
between these proteins appear to be regulated by the rab
GTPases because both p115 and GM130 can bind directly
to active rab1, and rab1 is required for the recruitment of
p115 to transport intermediates destined to fuse with the
cis-Golgi (Allan et al., 2000; Moyer et al., 2001; Weide et
al., 2001). A second GRASP complex is present on the
medial Golgi, comprising GRASP55 and the coiled-coil
protein golgin-45 (Short et al., 2001). This complex also
appears to be regulated by rabs because golgin-45 binds
directly to active rab2.
Several lines of evidence suggest that matrix proteins play
a key role in maintaining Golgi structure. Blocking the
Address correspondence to Martin Lowe, School of Biological Sciences,
University of Manchester, 2.205 Stopford Building, Oxford Road,
Manchester M13 9PT, UK. Tel.: 44-161-275-5387. Fax: 44-161-275-
5082. E-mail: lowe@man.ac.uk
Key words: Golgi apparatus; golgin; mitosis; Golgi structure; protein
phosphorylation
The Journal of Cell Biology
202 The Journal of Cell Biology
|
Volume 160, Number 2, 2003
interaction between p115 and GM130 in cells causes Golgi
vesicles to accumulate (Seemann et al., 2000b) and complete
removal of cellular p115 by antibody-induced degradation
leads to a highly vesiculated Golgi apparatus (Puthenveedu
and Linstedt, 2001). Depletion of golgin-45 has even more
dramatic effects, resulting in the complete loss of Golgi
structure and a redistribution of Golgi enzymes to the ER
(Short et al., 2001). Upon treatment of cells with brefeldin
A (BFA),* matrix proteins, unlike Golgi enzymes, remain
distinct from the ER and accumulate in cytoplasmic struc-
tures, often referred to as BFA remnants (Nakamura et al.,
1995; Seemann et al., 2000a). Upon removal of the drug,
these remnants can assemble into a structure reminiscent of
the Golgi ribbon even in the absence of other (nonmatrix)
Golgi proteins (Seemann et al., 2000a). These remnants can
be successfully partitioned into daughter cells during mitotic
division and nucleate post-mitotic Golgi assembly (Seemann
et al., 2002). Together, these results suggest that the matrix
proteins form a structural scaffold that can exist and divide
independently from Golgi enzyme–containing membranes.
However, this view has recently been challenged with the
finding that matrix proteins appear to constitutively cycle
between the Golgi apparatus and the ER or cytosol, suggest-
ing that the Golgi apparatus is a dynamic, steady-state sys-
tem that may be capable of self-assembly (Miles et al., 2001;
Ward et al., 2001). Recent work suggests that, at least in the
yeast
Pichia pastoris
, de novo assembly of the Golgi appara-
tus can occur (Bevis et al., 2002). Thus it is still unclear
whether a Golgi matrix exists, and if it does, whether such a
structure is a stable or highly dynamic one. Interestingly, it
has recently been shown that cis-Golgi matrix proteins can
also cycle into the late intermediate compartment, suggest-
ing that they might function in the incorporation of ER-
derived membranes into the Golgi apparatus (Marra et al.,
2001). Consistent with this, expression of a GM130 con-
struct defective in p115 binding reduced delivery of mem-
brane into the cis-Golgi.
During mitosis, membrane trafficking through the Golgi
apparatus is arrested and the Golgi undergoes extensive frag-
mentation (Warren et al., 1995; Lowe et al., 1998a). This
process is driven by protein phosphorylation, and, although
it is poorly understood at present, some progress has been
made in identifying the relevant kinases and their substrates.
CDK1-cyclin B is required for Golgi fragmentation in vitro
and one of its substrates has been identified as the cis-Golgi
matrix protein GM130 (Lowe et al., 1998b). CDK1-medi-
ated phosphorylation of GM130 on serine-25 abrogates
binding to p115, providing a molecular explanation for the
inhibition of vesicle docking seen in mitosis (Nakamura et
al., 1997; Lowe et al., 1998b). Mitogen-activated protein ki-
nase kinase I (MEK1) is also involved in mitotic fragmenta-
tion, but its effectors are not known (Acharya et al., 1998).
One possibility is that a monophosphorylated form of ERK
(pY-ERK), which has been localized to mitotic Golgi mem-
branes (Cha and Shapiro, 2001), or perhaps diphospho (ac-
tive) ERK, which can phosphorylate GRASP55 in vitro and
in vivo (Jesch et al., 2001), is involved. A third kinase that
appears to be required for mitotic fragmentation is Plk,
which can phosphorylate GRASP65 (Lin et al., 2000; Sut-
terlin et al., 2001).
We have used a proteomics-based strategy to identify
novel mitotic Golgi phosphoproteins with the reasoning
that these will include important structural proteins and/or
proteins involved in Golgi trafficking. Using this approach,
we identified the coiled-coil membrane protein golgin-84 as
a novel mitotic target and could show that this protein, al-
though not a component of the putative Golgi matrix, plays
an important role in the formation and maintenance of the
Golgi apparatus.
Results
Identification of golgin-84 as a mitotic phosphoprotein
To identify mitotic Golgi phosphoproteins, Golgi mem-
branes were incubated with interphase or mitotic cytosol in
the presence of [
-
32
P]ATP and analyzed by two-dimen-
sional (2D) 16-BAC/SDS-PAGE followed by autoradiogra-
phy. 20 spots were present exclusively in the mitotic sample
(Fig. 1 a). The previously known mitotic phosphoproteins
GM130 (Nakamura et al., 1997), GRASP65 (Barr et al.,
1997), GRASP55 (Jesch et al., 2001), and rab1 (Bailly et al.,
1991) were identified using a combination of mass spec-
trometry and Western blotting (Fig. 1 a; unpublished data).
To identify the other mitotic phosphoproteins, we decided
to first fractionate the membranes using sodium carbonate
extraction and Triton X-114 phase partitioning in order to
simplify the gel pattern before cutting out the spots and ana-
lyzing them by mass spectrometry. Analysis of the carbonate
pellet/Triton X-114 aqueous phase revealed that only one
mitotic phosphoprotein was present in this fraction (Fig. 1
b). This was identified using mass spectrometry as golgin-84,
a previously identified coiled-coil protein of unknown func-
tion, localized to the Golgi apparatus (Bascom et al., 1999).
To confirm that golgin-84 is phosphorylated specifically
in mitosis, polyclonal antibodies were used to immunopre-
cipitate golgin-84 from interphase or mitotically-treated
Golgi membranes. As shown in Fig. 1 c, golgin-84 was
highly phosphorylated by mitotic cytosol but only poorly by
interphase cytosol. Golgin-84 phosphorylation was stoichio-
metric because the mitotic form of the protein was shifted in
apparent molecular weight compared with the interphase
form upon SDS-PAGE (Fig. 1 d). Golgin-84 isolated from
mitotic HeLa cells was also shifted in apparent molecular
weight compared with that from interphase cells, confirming
that phosphorylation occurs in vivo (Fig. 1 d).
Localization of golgin-84 to the cis-Golgi network
Previous work has shown that golgin-84 is present on the
Golgi apparatus (Bascom et al., 1999), but the localization
of this protein at the ultrastructural level has not been ad-
dressed. To localize golgin-84 within the Golgi apparatus,
cryosections of A431 cells were labeled with polyclonal anti-
bodies to golgin-84 and examined under the electron micro-
scope. Golgin-84 was found predominantly on membranes
at the cis side of the Golgi stack (Fig. 2). Quantitation re-
*Abbreviations used in this paper: 2D, two dimensional; BFA, brefeldin
A; CGN, cis-Golgi network; NRK, normal rat kidney; RNAi, RNA in-
terference; siRNA, small interfering RNA; VSV-G, vesicular stomatitis
virus G protein.
The Journal of Cell Biology
Golgin-84 and Golgi ribbon formation |
Diao et al. 203
vealed that 34% of golgin-84 labeling was on cisternae
whereas 66% of labeling was on tubulo-vesicular profiles
(often referred to as the cis-Golgi network [CGN]) (Fig. 2
f). Of the tubulo-vesicular profile labeling, the vast majority
was on membranes at the lateral edges of the stack rather
then on membranes underlying the stacked cisternae (Fig. 2
f). Interestingly, golgin-84 labeling could frequently be de-
tected on tubulo-reticular elements apparently connecting
adjacent Golgi stacks (Fig. 2 d). A similar golgin-84 distri-
bution to that observed in A431 cells was also detected in
HeLa cells, suggesting that the localization of this protein is
not cell type dependent (Fig. 2 g).
The localization of golgin-84 to the cis side of the Golgi
apparatus is similar to that reported for the cis-Golgi matrix
protein GM130 (Nakamura et al., 1995; Marra et al.,
2001). We therefore analyzed the distribution of GM130 in
cryosections of A431 cells and compared it to that of golgin-
84. As shown in Fig. 2 e, strong labeling for GM130 was de-
tected along the face of the cis-most Golgi cisterna. Quanti-
tation revealed that
75% of GM130 labeling was present
on cisternae with only
25% on tubulo-vesicular profiles in
the vicinity of the Golgi stack (Fig. 2 f). Labeling of tubulo-
vesicular profiles was predominantly on membranes under-
lying the cisternae, with little GM130 detected on mem-
branes at the lateral edges of the stack. A similar GM130
distribution was also observed in HeLa cells (Fig. 2 g). Gol-
gin-84 and GM130 therefore localize to distinct regions of
the cis-Golgi, with golgin-84 more abundant at the lateral
edges of the Golgi stack while GM130 is present on more
central membranes.
Golgin-84 does not associate
with cis-Golgi matrix proteins
The different distributions of golgin-84 and GM130 in the
cis-Golgi suggested that golgin-84 may not be part of the pu-
tative Golgi matrix described by Seemann et al. (2000a,
Figure 1. Identification of golgin-84 as
a mitotic phosphoprotein. (a–c) Rat liver
Golgi membranes were incubated with
interphase or mitotic HeLa cytosol in the
presence of [-32P]ATP for 30 min at
30C and reisolated by centrifugation.
(a) Samples were subjected to 16-BAC/
SDS-PAGE 2D electrophoresis and
radiolabeled proteins were detected by
autoradiography. Positions of the known
mitotic phosphoproteins GM130,
GRASP65, GRASP55, and rab1 are
indicated. (b) The radiolabeled mem-
branes were washed with sodium
carbonate and the carbonate pellet was
extracted with Triton X-114. Proteins
partitioning into the aqueous phase were
analyzed by 2D 16-BAC/SDS-PAGE
and silver staining, followed by auto-
radiography. Arrows indicate the doublet
of spots corresponding to golgin-84.
The lower spot is a proteolytic cleavage
product. (c) The radiolabeled membranes
were solubilized in SDS and subjected
to immunoprecipitation with antibodies
to golgin-84. The membrane extracts
and immunoprecipitates were analyzed
by 1D SDS-PAGE followed by auto-
radiography. (d) Golgi membranes
incubated with interphase or mitotic
cytosol (in vitro) or total membrane
fractions prepared from interphase and
mitotic HeLa cells (in vivo) were analyzed
by 1D SDS-PAGE and immunoblotting
with antibodies to golgin-84.
The Journal of Cell Biology
204 The Journal of Cell Biology
|
Volume 160, Number 2, 2003
2002). We first tested whether golgin-84 can interact physi-
cally with cis-Golgi matrix proteins. Golgin-84 and the cis-
Golgi matrix proteins GM130 and p115 were immunopre-
cipitated from Golgi extracts under mild conditions and the
immunoprecipitates tested for the presence of golgin-84,
the matrix proteins GM130, p115, and GRASP65, and the
Golgi enzyme mannosidase I by immunoblotting. Even
though golgin-84 was efficiently precipitated by its antibody
(it was depleted from the unbound fraction), no matrix pro-
teins could be detected in the immunoprecipitate. Similarly,
no golgin-84 could be detected in the GM130 or p115 im-
munoprecipitates, which contained significant levels of
GM130, p115, and GRASP65 (Fig. 3 a). Thus, golgin-84
does not appear to physically interact with cis-Golgi matrix
proteins. To test more directly whether golgin-84 might exist
as part of the putative Golgi matrix, we studied its behavior
upon treatment of cells with BFA. As shown in Fig. 3 b, gol-
gin-84 redistributed to the ER in BFA-treated cells, as sug-
gested by previous work (Bascom et al., 1999). No golgin-84
was detected in the cytoplasmic GM130-containing punc-
tate structures, indicating that golgin-84 is not part of the
putative matrix described by Seemann et al. (2000a, 2002).
Golgi-84 is a binding partner for active rab1
Several Golgi-associated coiled-coil proteins interact with
the active or GTP-bound forms of the rab family of small
GTPases involved in membrane traffic. Specifically, p115, a
cis-Golgi vesicle tethering protein, binds to active rab1, and
GM130 binds to active rab1 and less efficiently to active
rab2, whereas golgin-45, a medial Golgi protein, binds only
Figure 2. Golgin-84 is localized to the
cis-Golgi network. (a–e) A431 cells were
processed for cryoelectron microscopy
and labeled with polyclonal antibodies
to golgin-84 (a–d) or GM130 (e) followed
by secondary antibodies coupled to
10-nm gold. Arrows indicate ER budding
profiles, and arrowheads indicate
golgin-84 (a–d) or GM130 (e) labeling.
(f–g) Quantitation of golgin-84 and
GM130 labeling of cryosections. The
relative number of gold particles over
Golgi cisternae, tubulo-vesicular profiles
lateral to the Golgi stack, tubulo-vesicular
profiles adjacent to the cis face of the
stack (central), or over other structures
was quantitated in gluteraldehyde-fixed
A431 cells (f) and paraformaldehyde-
fixed HeLa cells (g) as described in the
Materials and methods section. Note
that golgin-84 labeling is present on
the cis-most cisternae and more pre-
dominantly on tubulo-vesicular profiles
lateral to the stack, whereas GM130
labeling is mainly on the cis-most
cisternae of the stack (a–c). Bars, 200 nm.
The Journal of Cell Biology
Golgin-84 and Golgi ribbon formation |
Diao et al. 205
to active rab2 (Allan et al., 2000; Moyer et al., 2001; Short
et al., 2001; Weide et al., 2001). Because golgin-84 shares
similarities with these coiled-coil rab-binding proteins, we
analyzed whether this protein may itself be a novel rab effec-
tor. Golgi extract was incubated with immobilized rab pro-
teins in the inactive (GDP) or active (GTP
S) conformation
and bound proteins were analyzed by immunoblotting. As
expected, GM130 bound specifically to active rab1, but we
could not detect any binding to rab2 (Fig. 4 a). Rab2 did
however bind to golgin-45, as previously reported (Short et
al., 2001), demonstrating the functionality of this protein in
the binding assay (Fig. 4 a). Interestingly, golgin-84 bound
specifically to active rab1, with no detectable binding to
either rab2 or rab6. To confirm the interaction with rab1,
the yeast two-hybrid system was employed. As previously
shown, GM130 interacted directly with activated mutants
of rab1, rab2, and rab33b (Short et al., 2001; Valsdottir et
al., 2001; Weide et al., 2001) (Fig. 4 b). In contrast, golgin-
84 interacted only with activated rab1, and not with any of
the other rab proteins tested. Therefore, golgin-84 is a spe-
cific binding partner for active rab1. The rab1 binding site
Figure 3. Golgin-84 is not part of the Golgi matrix. (a) Rat liver Golgi extracts were immunoprecipitated under native conditions with antibodies
to either golgin-84, GM130, or p115, or with a control IgG. The immunoprecipitated (IP) and unbound fractions were analyzed by Western
blotting with antibodies to golgin-84, GM130, p115, GRASP65, and mannosidase I as indicated. (b) NRK cells were incubated with 5 g/ml
BFA for 1 h and then fixed and double labeled with antibodies to golgin-84 and GM130 or the ER marker calnexin. Bar, 10 m.
Figure 4.
Golgin-84 is a specific binding partner for rab1.
(a) GST-tagged rab1, rab2, and rab6 were loaded with GDP or
GTP
S and incubated with Golgi extract, and specifically eluted
proteins were analyzed by Western blotting with antibodies to
GM130, golgin-45, and golgin-84. (b) Full-length GM130 and
golgin-84 lacking the transmembrane domain were tested for inter-
action in the yeast two-hybrid system with the following rab proteins
carrying activating point mutations: rab1Q70L, rab2Q65L, rab5Q79L,
rab6Q72L, and rab33bQ92L. Interactions between the indicated
proteins results in growth on high selection medium. (c) Full-length
and truncation mutants of golgin-84 were tested for interaction with
rab1Q70L in the yeast two-hybrid system. The CT mutant corre-
sponds to the
Head
CC construct described in Fig. 5 without
the membrane anchor.
The Journal of Cell Biology
206 The Journal of Cell Biology
|
Volume 160, Number 2, 2003
was mapped using the two-hybrid system to the coiled-coil
region of golgin-84 (Fig. 4 c).
Overexpression or depletion of golgin-84
fragments the Golgi ribbon
The stiochiometric phosphorylation of golgin-84 in mitosis
together with its binding to active rab1 suggested that it may
play a role in Golgi structure and/or membrane trafficking
through the Golgi apparatus. To address a possible struc-
tural role for golgin-84, GFP-tagged full-length and trun-
cated versions of the protein were expressed in HeLa cells,
and effects upon Golgi structure were analyzed by immuno-
fluorescence microscopy (Fig. 5 a). At moderate expression
levels, none of the golgin-84 constructs elicited any signifi-
cant change in Golgi structure (unpublished data). Further-
more, constructs that failed to target to the Golgi apparatus
had no effects upon Golgi structure even at very high levels
of expression (Fig. 5 a; unpublished data). In contrast, ex-
pression at high levels of both full-length and golgin-84
lacking the head region had dramatic effects upon Golgi
structure, converting the ribbon into punctate structures dis-
persed throughout the cytoplasm (Fig. 5 b). EM analysis of
the Golgi fragments in cells overexpressing full-length gol-
gin-84 revealed that they are similar in overall organization
to the Golgi apparatus in control cells, comprising three to
four stacked cisternae and vesiculo-tubular profiles that label
heavily for the overexpressed protein (Fig. 5 c). Thus, over-
expression of golgin-84 does not lead to vesiculation of
Figure 5. Overexpression of golgin-84
fragments the Golgi ribbon. (a) Schematic
representation of the structure of golgin-
84 showing the constructs that were
expressed in HeLa cells. Each construct
was tagged at the NH2 terminus with
GFP. The predicted coiled-coil region
is shaded gray and the predicted trans-
membrane region is black. A summary
of the localization and effects upon
Golgi structure of each construct is
shown on the right. (b) HeLa cells
transfected with GFP-tagged WT (top),
Head (middle), and Head CC
(bottom) golgin-84 constructs were fixed
and stained with antibodies to GM130.
In the merged images on the right, DNA
is blue, GFP–golgin-84 is indicated in
green, GM130 is red, and yellow
indicates regions of overlap between
GFP–golgin-84 and GM130. Bar, 10 m.
(c) HeLa cells expressing GFP-tagged
WT golgin-84 were processed for cryo-
electron microscopy and labeled with
polyclonal antibodies to GFP followed
by rabbit anti–sheep antibodies and
protein A coupled to 8-nm gold.
Bars, 100 nm.
The Journal of Cell Biology
Golgin-84 and Golgi ribbon formation |
Diao et al. 207
Golgi stacks, just break-up of the ribbon. Fragmentation was
not due to the presence of GFP at the NH
2
terminus be-
cause identical results were obtained with golgin-84 con-
structs containing an NH
2
-terminal myc tag instead of GFP
(unpublished data). Fragmentation appeared more extensive
with the construct lacking the head region, suggesting that
the head may play some role in regulating Golgi structure.
Interestingly, golgin-84 lacking most of the coiled-coil re-
gion in addition to the head domain had only a minor effect
upon Golgi structure, even at extremely high expression lev-
els. Together, the results suggest that the coiled-coil domain
is important for the fragmentation observed, and that it is
only able to induce fragmentation when properly targeted to
the Golgi membranes.
To further examine the role of golgin-84 in maintaining
normal Golgi structure, we used small interfering RNA
(siRNA) to deplete cellular golgin-84 (Elbashir et al., 2001).
Transfection of HeLa cells with an siRNA duplex matching
the nucleotide sequence of golgin-84 resulted in a reduction
in golgin-84 levels of
90% after 3 d in culture (Fig. 6 a).
This reduction was specific because levels of GM130 were
unaffected, and transfection of cells with an siRNA duplex
targeting lamin A had no effect upon golgin-84 (or GM130)
levels. Immunofluorescence microscopy with antibodies to
GM130 showed that there was little effect upon Golgi mor-
phology in mock transfected cells or cells transfected with an
siRNA that effectively depleted cellular lamin A (Fig. 6 b).
Depletion of golgin-84, however, had a drastic effect upon
Golgi structure, breaking the ribbon into large fragments
dispersed in the cytoplasm (Fig. 6 b). The extent of Golgi
fragmentation correlated well with loss of golgin-84 over
time (Fig. 6 c). This, combined with the lack of fragmenta-
tion induced by prolonged exposure to the control lamin A
oligo, suggests that fragmentation is a consequence of gol-
gin-84 depletion and not cellular toxicity resulting from the
RNA interference (RNAi) treatment itself. Although pre-
dominantly punctate in appearance, the Golgi fragments oc-
casionally resembled short tubules. Fragments resulting from
golgin-84 depletion contained markers of the cis- (GM130),
medial (GalNacT2; Fig. 6 b), and trans-Golgi (TGN46; un-
published data), suggesting that some degree of Golgi orga-
nization may be retained in these cells. The Golgi fragments
did not extensively colocalize with the ER exit site marker
mSec23p, suggesting that they arise directly from the break-
up of the Golgi ribbon rather than the cycling through the
ER (Fig. 6 b).
To characterize the effects of golgin-84 depletion upon
Golgi morphology in more detail, siRNA-treated cells were
analyzed by electron microscopy. Interestingly, the Golgi
apparatus appeared significantly smaller in golgin-84–
depleted cells compared with control cells (Fig. 7, a and b).
Quantitation revealed that
75% of Golgi cisternae and
70% of Golgi tubulo-vesicular membranes were lost upon
golgin-84 depletion (Fig. 7 d). The overall morphology was
not, however, dramatically affected. The number of stacked
Figure 6.
Depletion of golgin-84 using RNAi fragments the Golgi
ribbon.
(a) HeLa cells were either mock transfected (no RNAi) or
transfected with duplex RNA to target lamin A or golgin-84 and,
after 1–4 d, were subjected to Western blotting with antibodies to
GM130 or golgin-84. (b) Mock transfected or RNAi-treated HeLa
cells were fixed and double labeled with antibodies to golgin-84,
lamin A, GalNacT2, mSec23p, and GM130. In the merged images
on the right, DNA is blue, golgin-84, lamin A, GalNacT2, mSec23p
are in green, and GM130 is red, with regions of overlap between
these proteins indicated in yellow. Bar, 10
m. (c) Quantitation of
golgin-84 levels and Golgi fragmentation in RNAi-treated cells.
Golgin-84 levels were measured by quantitating Western blots of
RNAi-treated HeLa cells as described in the Materials and methods
section. Golgi fragmentation was measured by immunofluorescence
microscopy using antibodies to GM130 to assess Golgi morphology.
For each time point, 200 cells were counted. The data are an average
of two independent experiments.
The Journal of Cell Biology
208 The Journal of Cell Biology
|
Volume 160, Number 2, 2003
cisternae per stack and the ratio of membrane in stacked cis-
ternae versus tubulo-vesicular profiles were similar in golgin-
84–depleted cells and control cells (unpublished data). De-
pletion of golgin-84 also had a marked effect upon the ER,
which was grossly exaggerated compared with control cells,
forming an elaborated network extending throughout the
cytoplasm (Fig. 7 c). The lumen of the ER often appeared
swollen with electron-dense material, suggesting that an ac-
cumulation of proteins had occurred there. Quantitation re-
vealed that the ER was
1.7 times larger in golgin-84–
depleted cells compared with control cells, while the nuclear
envelope was only marginally affected. Thus, depletion of
golgin-84 results in a dramatic loss of Golgi membranes and
a corresponding increase in the size of the ER.
Depletion of golgin-84 partially inhibits protein
transport from the ER to the cell surface
To study whether golgin-84 plays any role in protein traf-
ficking through the Golgi apparatus, we used a GFP-tagged
temperature-sensitive allele of the vesicular stomatitis virus G
protein (ts045 VSV-G). This well-characterized secretory
cargo marker accumulates in the ER at the nonpermissive
temperature of 39.5
C due to a reversible folding defect.
When shifted to the permissive temperature (31
C), cor-
rectly folded ts045G VSV-G is rapidly transported from the
ER, through the Golgi apparatus to the cell surface, where its
appearance can be monitored using an antibody against the
lumenal domain of the glycoprotein (Pepperkok et al., 1993;
Seemann et al., 2000b). As expected, VSV-G was efficiently
transported to the cell surface when control cells were shifted
to 31
C (Fig. 8 a). In cells depleted of golgin-84, VSV-G
could also be detected on the cell surface at 31
C (Fig. 8 a).
Golgin-84 depletion therefore does not block VSV-G trans-
port to the cell surface. To determine whether there is a more
subtle effect, transport of VSV-G to the cell surface was
quantitated in golgin-84–depleted cells relative to control
cells. This revealed that VSV-G transport to the cell surface
was inhibited by 15%, 39%, and 42% in golgin-84–depleted
cells after 30, 60, and 90 min at 31
C, respectively (Fig. 8 b).
These results suggest that golgin-84 is required for efficient
protein trafficking through the Golgi apparatus.
Discussion
In this report, we have identified golgin-84 as an important
structural component of the Golgi apparatus. Overexpression
of golgin-84 or depletion of the protein by RNAi results in ex-
tensive breakdown of the Golgi ribbon. The Golgi structures
formed upon golgin-84 depletion retain their stacked organi-
zation and contain Golgi resident proteins, but their overall
size is significantly smaller than that of a normal Golgi. This
suggests that golgin-84 is required for the incorporation of
membranes into the Golgi apparatus. This would be consis-
tent with the cis-Golgi network localization of golgin-84. This
highly pleiomorphic structure is where ER-derived transport
intermediates (vesiculo-tubular clusters or intermediate com-
partment) fuse and incorporate into the cis side of the Golgi
apparatus (Klumperman, 2000; Marra et al., 2001). This pro-
cess is at least partially dependent upon the cis-Golgi matrix
proteins GM130 and p115 because perturbation of the inter-
Figure 7. Golgin-84 depletion results in a significant loss of
membrane from the Golgi apparatus and an exaggerated ER.
Control (a) or golgin-84 RNAi–treated HeLa cells (b and c) were
fixed and embedded in Epon for electron microscopy. Large arrows
indicate Golgi membranes whereas small arrows indicate the ER.
Note the decrease in size of the Golgi apparatus and the exaggerated
and swollen ER in golgin-84–depleted cells. (d) Quantitation of the
amount of membrane in Golgi cisternae, Golgi vesicles, ER, and
nuclear envelope (NE) in golgin-84–depleted cells relative to control
cells expressed as density of membrane surface in cell volume. For
controls and RNAi samples, n 23 and 22 micrographs, respectively.
Bars, 200 nm.
The Journal of Cell Biology
Golgin-84 and Golgi ribbon formation |
Diao et al. 209
action between these proteins inhibits transport into the CGN
(Marra et al., 2001). Our results suggest that golgin-84 also
participates in the incorporation of membranes into the CGN.
How might golgin-84 function in building up the CGN?
At the structural level, golgin-84 is similar to the coiled-coil
proteins GM130, p115, giantin, and CASP, which have
been implicated in vesicle tethering at the Golgi apparatus
(Linstedt and Hauri, 1993; Nakamura et al., 1995; Sapper-
stein et al., 1995; Bascom et al., 1999; Gillingham et al.,
2002). In addition, we found that golgin-84 specifically in-
teracts with active rab1. Rab GTPases have an established
role in regulating membrane tethering in both the endocytic
and exocytic pathways (Waters and Hughson, 2000; Whyte
and Munro, 2002). We therefore believe that golgin-84 is a
tethering factor required for tethering incoming membranes
to the CGN and thereby promoting their fusion with this
compartment. Because golgin-84 is not part of the Golgi
matrix and is located to a region of the CGN devoid of the
matrix protein GM130, it is likely that golgin-84 partici-
pates in a tethering reaction different than that mediated by
the cis-Golgi matrix proteins.
What might this tethering reaction be? One possibility is
that golgin-84 tethers retrograde Golgi vesicles to the CGN.
In this case, depletion of golgin-84 would be expected to
cause accumulation of these vesicles, but no such accumula-
tion was observed in our experiments. Perhaps in the ab-
sence of tethering to the CGN, these vesicles would by
default fuse with the ER, causing an expansion of this
compartment as we have observed. However, we believe this
unlikely as no redistribution of Golgi enzymes, which have
been detected in Golgi-derived COPI vesicles (Lanoix et al.,
1999; Martinez-Menarguez et al., 2001), was detected in
our experiments. A more likely role for golgin-84 is in the
incorporation of incoming VTCs into the cis-Golgi. It could
either act in a parallel pathway to that mediated by the cis-
Golgi matrix proteins, or it may act at a temporally distinct
stage. Cis-Golgi matrix proteins cycle into the intermediate
compartment and are present on tubular connections be-
tween this pre-CGN compartment and the CGN itself
(Marra et al., 2001). Nearly all (
85%) of these GM130-
containing structures label for the cargo protein VSV-G,
suggesting that cis-Golgi matrix proteins mediate the first
step in the incorporation of VTCs into the CGN (Marra et
al., 2001). We could not detect any golgin-84 in the inter-
mediate compartment under steady-state conditions or upon
incubation at 15
C (unpublished data), suggesting that gol-
gin-84 is not involved at such an early step. We therefore
think it likely that golgin-84 operates after the cis-Golgi ma-
trix proteins. The current model we favor is that golgin-84
tethers newly-forming cis-Golgi matrix-positive CGN ele-
ments and promotes their lateral fusion, which may be a ho-
motypic event. The presence of golgin-84 at the rims of
CGN elements places it in the ideal position for connecting
these together laterally and promoting their fusion to form a
continuous cisternal/ribbon structure.
In addition to the cis-Golgi matrix proteins and golgin-84,
the cis-Golgi also contains the multisubunit tethering com-
plexes TRAPP1, TRAPPII, and COG (for review see Whyte
and Munro, 2002). Why have so many tethering complexes
on one compartment? One reason may be that the cis-Golgi
participates in multiple transport pathways. It receives mem-
brane from the ER and from retrograde Golgi vesicles, and
exports membrane back to the ER as well as forward into the
Golgi stack. Thus, different tethering factors may be re-
quired for different transport steps. For example, COG ap-
pears to be required for the tethering of retrograde Golgi ves-
icles (Suvorova et al., 2002), whereas TRAPP1 is required for
the tethering of anterograde ER-derived vesicles (Sacher et
al., 2001). Another reason may be that the CGN is where
Golgi cisternae are formed. The transition from pleiomor-
phic tubulo-vesicular clusters into a regular array of flattened
and stacked cisternae is likely to involve multiple tethering
and fusion events, with different proteins responsible for dif-
ferent reactions in the pathway. Rab1 (Ypt1 in yeast) may be
Figure 8. Depletion of golgin-84 partially inhibits transport of
VSV-G from the ER to the cell surface. Control or golgin-84
RNAi–treated HeLa cells were transfected with a plasmid encoding
GFP-tagged ts045G VSV-G protein. Cells were incubated at 39.5C
to arrest ts045G VSV-G in the ER, and then chased at 31C for
various times to allow transport before fixation and labeling for cell
surface VSV-G. (a) An example of cells shifted to 31C for 60 min
and labeled for cell surface VSV-G. (b) The extent of VSV-G transport
was measured as indicated in the Materials and methods and is
expressed as the ratio of cell surface to total VSV-G fluorescence.
The data shown are representative of two experiments with n 15
for all data points in each experiment.
The Journal of Cell Biology
210 The Journal of Cell Biology
|
Volume 160, Number 2, 2003
a master regulator of this pathway, as it has been shown to
interact with all of the cis-Golgi–associated tethering com-
plexes (Whyte and Munro, 2002).
It was recently reported that the Golgi matrix proteins
alone are sufficient to form a perinuclear Golgi-like ribbon
(Seemann et al., 2000a). However, at the EM level, this
structure was comprised predominantly of vesicles rather
than cisternae (Seemann et al., 2000a), suggesting that addi-
tional factors are required for cisternae formation and Golgi
apparatus assembly. One of these factors may be golgin-84,
which would be expected to be absent from the matrix-con-
taining structures described by Seemann et al. (2000a). This
would be consistent with the predicted role of golgin-84 in
promoting lateral fusion of Golgi membranes.
We found that protein transport was inhibited by only
40% in cells depleted of golgin-84. There are several possi-
ble explanations for this. One possibility is that golgin-84 is
essential for transport, but the residual protein remaining af-
ter depletion, corresponding to only a few percent of the
normal amount, is sufficient for transport to occur. Alter-
natively, golgin-84 may have no direct role in transport,
with the transport inhibition merely reflecting the reduced
amount of Golgi membranes present in golgin-84–depleted
cells. In this case, golgin-84 would function purely as a struc-
tural protein. Finally, golgin-84 may improve the efficiency
of transport without actually being essential for the process
per se. This would be analogous to the situation with
GM130 and p115. Blocking the interaction between these
tethering proteins only partially inhibits protein transport
through the Golgi apparatus (Seemann et al., 2000b; Marra
et al., 2001). Currently, we cannot distinguish between these
possibilities, and further experiments will be required to fully
understand the role of golgin-84 in protein transport.
Golgin-84 is the second nonmatrix protein, after rab1
(Bailly et al., 1991), to be identified as a target for mitotic
kinases. Further work is required to elucidate the role of gol-
gin-84 phosphorylation and to determine whether this plays
a part in the mitotic fragmentation process. One possibility
is that phosphorylation is required in the early stages of mi-
tosis when the Golgi ribbon is broken down to mini-stacks
arranged around the nuclear envelope (Misteli and Warren,
1995; Shima et al., 1998). This would be most consistent
with the predicted role of golgin-84 in linking membranes
into a ribbon. Interestingly, there are no evolutionarily con-
served candidate MAPK or CDK1 phosphorylation sites in
golgin-84, suggesting that it is either a substrate for Plk (Sut-
terlin et al., 2001) or another kinase not known to be in-
volved in the fragmentation process.
In summary, we have identified golgin-84 as a novel Golgi
structural protein. The challenge now is to identify the mo-
lecular interactions of golgin-84 during both interphase and
mitosis. This should not only lead to a greater understanding
of Golgi apparatus assembly and maintenance but also illumi-
nate how these processes are regulated during the cell cycle.
Materials and methods
Antibodies
Polyclonal antibodies to golgin-84 were raised in sheep using GST-tagged
golgin-84 head or coiled-coil domain as immunogens. Antibodies were ad-
sorbed against GST and affinity-purified against the corresponding fusion
proteins covalently coupled to glutathione-sepharose (Amersham Bio-
sciences). Polyclonal antibodies were raised in sheep against GST-tagged
GFP and affinity-purified against the same protein coupled to glutathione-
sepharose. The following antibodies were also used in this study: 4H1
monoclonal anti-p115; MLO7 (anti-N73pep) polyclonal anti-GM130 (Na-
kamura et al., 1997); 4A3 monoclonal anti-GM130 (Seemann et al., 2002);
polyclonal anti-GM130 for electron microscopy (from Maria Antonietta De
Matteis, Mario-Negri Institute, Santa Maria Imbaro, Italy); rabbit polyclonal
antibodies to mannosidase I, GRASP65, and golgin-45 (from Francis Barr,
Max-Planck Institute for Biochemistry, Martinsried, Germany); monoclonal
anti-GalNacT2 (from Dr. Henrik Clausen, University of Copenhagen, Den-
mark); polyclonal anti-calnexin (from Dr. Stephen High, University of
Manchester, Manchester, UK); and monoclonal anti–VSV-G lumenal do-
main (from Dr. Rainer Pepperkok, European Molecular Biology Laboratory,
Heidelberg, Germany). Goat polyclonal antibodies to lamin A/C (N-18)
were purchased from Santa Cruz Biotechnology, Inc. Rabbit polyclonal
anti-mSec23p antibodies were purchased from Affinity BioReagents, Inc.
Fluorophore and HRP-conjugated secondary antibodies were purchased
from Molecular Probes and Tago Immunologicals, respectively.
In vitro phosphorylation of Golgi membranes
and 16-BAC gel electrophoresis
Rat liver Golgi membranes were purified as described previously (Hui et al.,
1998). Interphase and mitotic cytosols were prepared from spinner HeLa
cells according to Sönnichsen et al. (1996) and desalted into buffer A (20
mM
-glycerophosphate, 15 mM EGTA, 50 mM KOAc, 10 mM MgOAc, 2
mM ATP, 1 mM DTT, 0.2 M sucrose). Golgi membranes were incubated
with desalted interphase and mitotic HeLa cytosol (9 mg/ml) in the presence
of 0.2
Ci/
l [
-
32
P]ATP for 30 min at 30
C. The incubated membranes
were then adjusted to 1.6 M sucrose, overlaid with 1.2 M sucrose, 1.0 M
sucrose, and finally 0.4 M sucrose (all sucrose solutions were made in TKN
buffer [20 mM Tris-Cl, pH 7.4, 0.1 M KCl, 0.1 M NaF and 1 mM DTT]), and
centrifuged for 4 h at 55,000 rpm in a SW55 rotor. The Golgi membranes
(at the 0.4 M/1.0 M interface) were collected and pelleted by centrifugation
at 55,000 rpm for 30 min in a TLA55 rotor. The membranes were either sol-
ubilized directly into sample buffer and subjected to 2D 16-BAC/SDS-PAGE
(Hartinger et al., 1996) or washed with 0.2 M sodium carbonate and the
carbonate pellet was further extracted with 1% Triton X-114 (Nakamura et
al., 1995) before electrophoresis. 16-BAC/SDS-PAGE gels were analyzed by
silver staining and autoradiography.
Mass spectrometry
Radiolabeled proteins were excised from dried 2D gels with a protein-
free razor blade. Excised spots were subjected to in-gel digestion with
trypsin and the resulting peptides were analyzed using a MALDI-TOF in-
strument (M@LDI; Micromass) and probability-based database searching
(Pappin, 2003). To confirm the identity of a protein, the digest extracts
were analyzed by nano electrospray on an ion trap instrument (Finnigan
LCQ Deca; Thermoquest). MS/MS data were obtained for a number of
peptides and the spectra were used to query the MS/MS Ion Search pro-
gram on MASCOT.
Immunoprecipitation of golgin-84
For analysis of golgin-84 phosphorylation,
32
P-labeled Golgi membranes
were resuspended in TKN buffer containing 1% SDS and protease inhibi-
tors, boiled for 3 min, mixed with an equal volume of ice-cold 4% Triton
X-100, and clarified by centrifugation at 14,000 rpm for 10 min. 2
g of
polyclonal antibodies to golgin-84 and 10
l protein G–sepharose beads
were added and incubated at 4
C for 2–4 h at 4
C. After washing three
times with IP buffer (TKN containing 0.5% TX-100), bound proteins were
eluted by boiling in SDS sample buffer and analyzed by 1D SDS-PAGE fol-
lowed by autoradiography. To test for coimmunoprecipitation of golgin-84
and matrix proteins, Golgi membranes were extracted in IP buffer lacking
NaF for 30 min on ice, clarified, and incubated with 2
g of anti–golgin-
84, anti-GM130 (4A3), or anti-p115 (4H1) antibodies and 10
l protein
G–sepharose. Beads were washed with IP buffer lacking NaF, boiled in
SDS sample buffer, and eluted proteins were analyzed by 1D SDS-PAGE
and immunoblotting with the appropriate antibodies.
Rab effector binding assay
Binding of Golgi proteins to GST-tagged rab proteins was performed ac-
cording to Short et al. (2001) except that 0.25 mg rab protein and 25
l
glutathione-sepharose beads were incubated with 100
g Golgi extract in
a final volume of 200
l for 2 h at 4
C. After elution, bound proteins were
precipitated with 10% TCA and analyzed by Western blotting.
The Journal of Cell Biology
Golgin-84 and Golgi ribbon formation |
Diao et al. 211
Molecular biology and yeast two-hybrid analysis
Standard molecular biology techniques were used for all constructs;
primer sequences are available upon request. The full-length and trun-
cated versions of human golgin-84 cDNA were inserted into the BglII and
EcoRI sites of the pEGFP-C1 vector (CLONTECH Laboratories, Inc.) or the
BamHI and EcoRI sites of a modified pcDNA3.1 vector (Stratagene) con-
taining an NH
2
-terminal myc tag. Full-length and truncated versions of hu-
man golgin-84 cDNA lacking the trans-membrane domain were inserted
into the yeast two-hybrid activation domain vector pGADT7. All bait vec-
tor pGBT9/rab GTPase constructs were provided by Francis Barr. The
pGADT7/golgin-84 and pGBT9/rab GTPase plasmids were cotransformed
into the yeast reporter strain AH109 on synthetic medium lacking leucine
and tryptophan (low selection) and then restreaked onto synthetic medium
lacking leucine, tryptophan, histidine, and adenine with 2% glucose as the
carbon source (high selection) according to the CLONTECH Laboratories,
Inc. yeast protocol handbook.
Cell culture and drug treatments
HeLa, normal rat kidney (NRK), and A431 cells were cultured at 37
C and
5% CO
2
in DME containing 10% FCS. NRK cells were incubated with 5
g/ml BFA in tissue culture medium for 1 h at 37
C before fixation.
Transfections and RNAi
HeLa cells were transfected with DNA plasmids using Fugene 6 (Roche
Biochemicals) according to the manufacturer’s instructions. RNAi was per-
formed on HeLa cells using oligofectamine (Life Technologies) with duplex
RNA oligos (Dharmacon Research) for 1–4 d as described by Elbashir et al.
(2001). Golgin-84 was targeted with the sequence AAGTAGGATCTCG-
GACACCAG and the lamin A control was described previously (Elbashir et
al., 2001). Golgin-84 levels were quantitated from Western blots accord-
ing to Sönnichsen et al. (1998).
Fluorescence microscopy
Cells were grown on coverslips and fixed in 100% methanol at
20C for
4 min. Coverslips were incubated with primary antibodies diluted into PBS
containing 0.5 mg/ml BSA for 20 min at RT, washed, and incubated with
PBS/BSA containing fluorophore-conjugated secondary antibodies and
200 ng/ml Hoechst 33342 for an additional 20 min at RT. Coverslips were
mounted in Mowiol and analyzed by conventional epifluorescence mi-
croscopy using an Olympus BX60 upright microscope with a MicroMax
CCD camera (Roper Scientific) driven by Metamorph software. Confocal
images were obtained using a Leica NT confocal microscope. All images
are projections of optocal sections in the z axis at 0.5-m intervals.
VSV-G transport assays
3 d after RNAi treatment, HeLa cells were transfected with a plasmid en-
coding GFP-tagged ts045G VSV-G (provided by Patrick Keller, Max-
Planck Institute for Molecular Cell Biology and Genetics, Dresden, Ger-
many) for 1 h at 37C and then 12 h at 39.5C. Cells were then incubated
at 4C for 30 min to promote VSV-G protein folding, the growth medium
was replaced with prewarmed medium (31C), and the cells were incu-
bated for a further 0, 30, 60, or 90 min at 31C. Cells were then fixed in
3.5% paraformaldehyde and cell surface VSV-G was detected with a
monoclonal antibody to the VSV-G lumenal domain and a Texas red–con-
jugated anti–mouse secondary antibody and total VSV-G by GFP. The ratio
of surface to total measured fluorescence was used to calculate the extent
of VSV-G protein transport (Pepperkok et al., 1993; Seemann et al.,
2000b).
Electron microscopy
Cells were fixed in 2% gluteraldehyde or 8% paraformaldehyde and pro-
cessed for cryosectioning and immunogold labeling as described by Far-
maki et al. (1999), except the sections were picked up using the modified
pick up method (Liou et al., 1996); one part methyl cellulose and three
parts 2.3 M sucrose in PBS. For quantitation of gold labeling, cell profiles
contained in a randomly selected grid square were scanned systematically
and Golgi areas were identified by the presence of cisternal stacks and/or
vesicular profiles labeled for golgin-84 or GM130. Labeling was assigned
to one of four categories: Golgi cisternae, tubulo-vesicular profiles lateral
to the Golgi stack, tubulo-vesicular profiles on the cis face of the stack, and
any other labeling detected. Golgi cisternae are defined as membrane-
bounded profiles with a length/breadth ratio of 4 or more. Tubulo-vesicu-
lar profiles are noncisternal profiles with a diameter of 80 nm. The lateral
aspect of the stack was delineated by drawing a line across the end of the
stack orthogonal to the cisternae. For structural and quantitative analysis of
RNAi-treated cells, samples were fixed in 2% gluteraldehyde, post-fixed
with reduced osmium tetroxide, and embedded in epoxy resin according
to Lucocq et al. (1989). The surface density of membranes in the cell were
estimated using stereological methods from the formula 2I/L, in which I
represents intersections of the lines on a square lattice grid with the mem-
brane of interest and L is the total line length applied to the reference
space (Lucocq, 1993).
We thank Drs. Francis Barr, Henrik Clausen, Maria Antonietta De Matteis,
Viki Allan, Stephen High, Rainer Pepperkok, and Patrick Keller for gener-
ously providing antibodies and reagents as noted above. We are grateful to
Joanna Woodburn for preparing the GFP antibodies and Drs. Philip Wood-
man and Viki Allan for critical reading of the manuscript.
This work was supported by a Medical Research Council Career Devel-
opment Award to M. Lowe (G120/483).
Submitted: 8 July 2002
Revised: 3 December 2002
Accepted: 3 December 2002
References
Acharya, U., A. Mallabiabarrena, J.K. Acharya, and V. Malhotra. 1998. Signaling
via mitogen-activated protein-kinase kinase (MEK 1) is required for Golgi
fragmentation during mitosis. Cell. 92:183–192.
Allan, B.B., B.D. Moyer, and W.E. Balch. 2000. Rab1 recruitment of p115 into a
cis-SNARE complex: programming budding COPII vesicles for fusion. Sci-
ence. 289:444–448.
Bailly, E., M. McCaffrey, N. Touchot, A. Zahraoui, B. Goud, and M. Bornens.
1991. Phosphorylation of two small GTP-binding proteins of the Rab fam-
ily by p34cdc2. Nature. 350:715–718.
Barr, F.A., M. Puype, J. Vandekerckhove, and G. Warren. 1997. GRASP65, a pro-
tein involved in the stacking of Golgi cisternae. Cell. 91:253–262.
Barr, F.A., N. Nakamura, and G. Warren. 1998. Mapping the interaction between
GRASP65 and GM130, components of a protein complex involved in the
stacking of Golgi cisternae. EMBO J. 17:3258–3268.
Bascom, R.A., S. Srinivasan, and R.L. Nussbaum. 1999. Identification and charac-
terization of golgin-84, a novel Golgi integral membrane protein with a cy-
toplasmic coiled-coil domain. J. Biol. Chem. 274:2953–2962.
Bevis, B.J., A.T. Hammond, C.A. Reinke, and B.S. Glick. 2002. De novo forma-
tion of transitional ER sites and Golgi structures in Pichia pastoris. Nat. Cell
Biol. 4:750–756.
Cha, H., and P. Shapiro. 2001. Tyrosine-phosphorylated extracellular signal–regu-
lated kinase associates with the Golgi complex during G2/M phase of the
cell cycle: evidence for regulation of Golgi structure. J. Cell Biol. 153:1355–
1367.
De Matteis, M.A., and J.S. Morrow. 2000. Spectrin tethers and mesh in the bio-
synthetic pathway. J. Cell Sci. 113:2331–2343.
Elbashir, S.M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl.
2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cul-
tured mammalian cells. Nature. 411:494–498.
Farmaki, T., S. Ponnambalam, A.R. Prescott, H. Clausen, B.L. Tang, W. Hong,
and J.M. Lucocq. 1999. Forward and retrograde trafficking in mitotic ani-
mal cells. ER-Golgi transport arrest restricts protein export from the ER into
COPII-coated structures. J. Cell Sci. 112:589–600.
Gillingham, A.K., A.C. Pfeifer, and S. Munro. 2002. CASP, the alternatively
spliced product of the gene encoding the CCAAT-displacement protein
transcription factor, is a Golgi membrane protein related to giantin. Mol.
Biol. Cell. 13:3761–3774.
Hartinger, J., K. Stenius, D. Hogemann, and R. Jahn. 1996. 16-BAC/SDS-PAGE:
a two-dimensional gel electrophoresis system suitable for the separation of
integral membrane proteins. Anal. Biochem. 240:126–133.
Hui, N., N. Nakamura, P. Slusarewicz, and G. Warren. 1998. Purification of rat
liver Golgi stacks. In Cell Biology: A Laboratory Handbook. Vol. 2. J. Celis,
editor. Academic Press Inc., Orlando, FL. 46–55.
Jesch, S.A., T.S. Lewis, N.G. Ahn, and A.D. Linstedt. 2001. Mitotic phosphoryla-
tion of Golgi reassembly stacking protein 55 by mitogen-activated protein
kinase ERK2. Mol. Biol. Cell. 12:1811–1817.
Klumperman, J. 2000. Transport between ER and Golgi. Curr. Opin. Cell Biol. 12:
445–449.
Lanoix, J., J. Ouwendijk, C.C. Lin, A. Stark, H.D. Love, J. Ostermann, and T.
Nilsson. 1999. GTP hydrolysis by arf-1 mediates sorting and concentration
The Journal of Cell Biology
212 The Journal of Cell Biology | Volume 160, Number 2, 2003
of Golgi resident enzymes into functional COP I vesicles. EMBO J. 18:
4935–4948.
Lin, C.Y., M.L. Madsen, F.R. Yarm, Y.J. Jang, X. Liu, and R.L. Erikson. 2000. Pe-
ripheral Golgi protein GRASP65 is a target of mitotic polo-like kinase (Plk)
and Cdc2. Proc. Natl. Acad. Sci. USA. 97:12589–12594.
Linstedt, A.D., and H.P. Hauri. 1993. Giantin, a novel conserved Golgi mem-
brane protein containing a cytoplasmic domain of at least 350 kDa. Mol.
Biol. Cell. 4:679–693.
Liou, W., H.J. Geuze, and J.W. Slot. 1996. Improving structural integrity of cryo-
sections for immunogold labeling. Histochem. Cell Biol. 106:41–58.
Lowe, M., N. Nakamura, and G. Warren. 1998a. Golgi division and membrane
traffic. Trends Cell Biol. 8:40–44.
Lowe, M., C. Rabouille, N. Nakamura, R. Watson, M. Jackman, E. Jamsa, D.
Rahman, D.J. Pappin, and G. Warren. 1998b. Cdc2 kinase directly phos-
phorylates the cis-Golgi matrix protein GM130 and is required for Golgi
fragmentation in mitosis. Cell. 94:783–793.
Lucocq, J.M. 1993. Unbiased 3-D quantitation of ultrastructure in cell biology.
Trends Cell Biol. 3:345–358.
Lucocq, J.M., E.G. Berger, and G. Warren. 1989. Mitotic Golgi fragments in
HeLa cells and their role in the reassembly pathway. J. Cell Biol. 109:463–
474.
Marra, P., T. Maffucci, T. Daniele, G.D. Tullio, Y. Ikehara, E.K. Chan, A. Luini,
G. Beznoussenko, A. Mironov, and M.A. De Matteis. 2001. The GM130
and GRASP65 Golgi proteins cycle through and define a subdomain of the
intermediate compartment. Nat. Cell Biol. 3:1101–1113.
Martinez-Menarguez, J.A., R. Prekeris, V.M. Oorschot, R. Scheller, J.W. Slot, H.J.
Geuze, and J. Klumperman. 2001. Peri-Golgi vesicles contain retrograde but
not anterograde proteins consistent with the cisternal progression model of
intra-Golgi transport. J. Cell Biol. 155:1213–1224.
Miles, S., H. McManus, K.E. Forsten, and B. Storrie. 2001. Evidence that the en-
tire Golgi apparatus cycles in interphase HeLa cells: sensitivity of Golgi ma-
trix proteins to an ER exit block. J. Cell Biol. 155:543–556.
Misteli, T., and G. Warren. 1995. Mitotic disassembly of the Golgi apparatus in
vivo. J. Cell Sci. 108:2715–2727.
Moyer, B.D., B.B. Allan, and W.E. Balch. 2001. Rab1 interaction with a GM130
effector complex regulates COPII vesicle cis-Golgi tethering. Traffic. 2:268–
276.
Nakamura, N., C. Rabouille, R. Watson, T. Nilsson, N. Hui, P. Slusarewicz, T.E.
Kreis, and G. Warren. 1995. Characterization of a cis-Golgi matrix protein,
GM130. J. Cell Biol. 131:1715–1726.
Nakamura, N., M. Lowe, T.P. Levine, C. Rabouille, and G. Warren. 1997. The
vesicle docking protein p115 binds GM130, a cis-Golgi matrix protein, in a
mitotically regulated manner. Cell. 89:445–455.
Nelson, D.S., C. Alvarez, Y.S. Gao, R. Garcia-Mata, E. Fialkowski, and E. Sztul.
1998. The membrane transport factor TAP/p115 cycles between the Golgi
and earlier secretory compartments and contains distinct domains required
for its localization and function. J. Cell Biol. 143:319–331.
Pappin, D.J. 2003. Peptide mass fingerprinting using MALDI-TOF mass spec-
trometry. Methods Mol. Biol. 211:211–219.
Pepperkok, R., J. Scheel, H. Horstmann, H.P. Hauri, G. Griffiths, and T.E. Kreis.
1993. -COP is essential for biosynthetic membrane transport from the en-
doplasmic reticulum to the Golgi complex in vivo. Cell. 74:71–82.
Pfeffer, S.R. 2001. Constructing a Golgi complex. J. Cell Biol. 155:873–875.
Puthenveedu, M.A., and A.D. Linstedt. 2001. Evidence that Golgi structure de-
pends on a p115 activity that is independent of the vesicle tether compo-
nents giantin and GM130. J. Cell Biol. 155:227–238.
Rambourg, A., and Y. Clermont. 1997. Three-dimensional structure of the Golgi
apparatus in mammalian cells. In The Golgi Apparatus. E.G. Berger and J.
Roth, editors. Birkhauser Verlag, Basel, Switzerland. 37–61.
Sacher, M., J. Barrowman, W. Wang, J. Horecka, Y. Zhang, M. Pypaert, and S.
Ferro-Novick. 2001. TRAPP I implicated in the specificity of tethering in
ER-to-Golgi transport. Mol. Cell. 7:433–442.
Sapperstein, S.K., D.M. Walter, A.R. Grosvenor, J.E. Heuser, and M.G. Waters.
1995. p115 is a general vesicular transport factor related to the yeast endo-
plasmic reticulum to Golgi transport factor Uso1p. Proc. Natl. Acad. Sci.
USA. 92:522–526.
Seemann, J., E. Jokitalo, M. Pypaert, and G. Warren. 2000a. Matrix proteins can
generate the higher order architecture of the Golgi apparatus. Nature. 407:
1022–1026.
Seemann, J., E.J. Jokitalo, and G. Warren. 2000b. The role of the tethering pro-
teins p115 and GM130 in transport through the Golgi apparatus in vivo.
Mol. Biol. Cell. 11:635–645.
Seemann, J., M. Pypaert, T. Taguchi, J. Malsam, and G. Warren. 2002. Partition-
ing of the matrix fraction of the Golgi apparatus during mitosis in animal
cells. Science. 295:848–851.
Shima, D.T., N. Cabrera-Poch, R. Pepperkok, and G. Warren. 1998. An ordered
inheritance strategy for the Golgi apparatus: visualization of mitotic disas-
sembly reveals a role for the mitotic spindle. J. Cell Biol. 141:955–966.
Short, B., C. Preisinger, R. Korner, R. Kopajtich, O. Byron, and F.A. Barr. 2001.
A GRASP55–rab2 effector complex linking Golgi structure to membrane
traffic. J. Cell Biol. 155:877–883.
Shorter, J., and G. Warren. 1999. A role for the vesicle tethering protein, p115, in
the post-mitotic stacking of reassembling Golgi cisternae in a cell-free sys-
tem. J. Cell Biol. 146:57–70.
Slusarewicz, P., T. Nilsson, N. Hui, R. Watson, and G. Warren. 1994. Isolation of
a matrix that binds medial Golgi enzymes. J. Cell Biol. 124:405–413.
Sönnichsen, B., R. Watson, H. Clausen, T. Misteli, and G. Warren. 1996. Sorting
by COP I–coated vesicles under interphase and mitotic conditions. J. Cell
Biol. 134:1411–1425.
Sönnichsen, B., M. Lowe, T. Levine, E. Jämsä, B. Dirac-Svejstrup, and G. Warren.
1998. A role for giantin in docking COPI vesicles to Golgi membranes. J.
Cell Biol. 140:1013–1023.
Sutterlin, C., C.Y. Lin, Y. Feng, D.K. Ferris, R.L. Erikson, and V. Malhotra. 2001.
Polo-like kinase is required for the fragmentation of pericentriolar Golgi
stacks during mitosis. Proc. Natl. Acad. Sci. USA. 98:9128–9132.
Suvorova, E.S., R. Duden, and V.V. Lupashin. 2002. The Sec34/Sec35p complex,
a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts
with Golgi SNAREs and COPI vesicle coat proteins. J. Cell Biol. 157:631–
643.
Valsdottir, R., H. Hashimoto, K. Ashman, T. Koda, B. Storrie, and T. Nilsson.
2001. Identification of rabaptin-5, rabex-5, and GM130 as putative effectors
of rab33b, a regulator of retrograde traffic between the Golgi apparatus and
ER. FEBS Lett. 508:201–209.
Ward, T.H., R.S. Polishchuk, S. Caplan, K. Hirschberg, and J. Lippincott-
Schwartz. 2001. Maintenance of Golgi structure and function depends on
the integrity of ER export. J. Cell Biol. 155:557–570.
Warren, G., T. Levine, and T. Misteli. 1995. Mitotic disassembly of the mamma-
lian Golgi apparatus. Trends Cell Biol. 5:413–416.
Waters, M.G., and F.M. Hughson. 2000. Membrane tethering and fusion in the
secretory and endocytic pathways. Traffic. 1:588–597.
Weide, T., M. Bayer, M. Koster, J.P. Siebrasse, R. Peters, and A. Barnekow. 2001.
The Golgi matrix protein GM130: a specific interacting partner of the small
GTPase rab1b. EMBO Rep. 2:336–341.
Whyte, J.R.C., and S. Munro. 2002. Vesicle tethering complexes in membrane
traffic. J. Cell Sci. 115:2627–2637.
