Download The Sec34/35 Golgi Transport Complex Is Related to the Exocyst

Developmental Cell, Vol. 1, 527–537, October, 2001, Copyright 2001 by Cell Press
The Sec34/35 Golgi Transport Complex Is Related
to the Exocyst, Defining a Family of Complexes
Involved in Multiple Steps of Membrane Traffic
James R.C. Whyte and Sean Munro1
MRC Laboratory of Molecular Biology
Hills Road
Cambridge CB2 2QH
United Kingdom
Summary
The specificity of intracellular vesicle transport is mediated in part by tethering factors that attach the vesicle to the destination organelle prior to fusion. We
have identified a protein, Dor1p, that is involved in
vesicle targeting to the yeast Golgi apparatus and
found it to be associated with seven further proteins.
Identification of these revealed that they include
Sec34p and Sec35p, the two known components of
the Sec34/35 complex previously proposed to tether
vesicles to the Golgi. Of the six previously uncharacterized components, four have homologs in higher eukaryotes, including a subunit of a mammalian Golgi
transport complex. Furthermore, several of the proteins show distant homology to components of two
other putative tethering complexes, the exocyst and
the Vps52/53/54 complex, revealing that tethering factors involved in different membrane traffic steps are
structurally related.
Introduction
Transport of proteins and lipids within the secretory
pathway of eukaryotic cells requires the formation of
carriers or vesicles that bud from one organelle and then
deliver their cargo to the correct target compartment.
This requires that the vesicle specifically recognizes and
then fuses with only one of the many membranes within
the cytoplasm. The process of fusion itself is mediated
by the SNARE proteins, membrane-anchored coiled-coil
proteins present on both vesicle and target organelle
(Söllner et al., 1993). During the vesicle fusion process,
specific SNAREs assemble into a four-helix bundle
whose formation serves to bring the vesicle and target
membranes into close proximity (Sutton et al., 1998).
The degree of specificity imparted by the SNAREs has
been a matter of some debate (Chen and Scheller, 2001;
McNew et al., 2000; Pelham, 2001). Although there are
specific sets of SNARE proteins associated with each
transport step, it is now clear that while they may contribute to the fidelity of fusion between membranes,
other factors must also play a role. For several transport
steps it has been possible to identify “tethering factors”
that are required for vesicle delivery but appear to function upstream of membrane fusion (Pfeffer, 1999; Waters
and Hughson, 2000). How these factors act is still being
elucidated, but those identified are soluble proteins or
complexes that often bind specifically to a single target
organelle or class of vesicle, and in some cases have
1
Correspondence: [email protected]
been shown to be necessary for attachment of the incoming transport vesicles to the correct organelle. Thus,
tethering may represent the earliest stage at which specificity is conferred on a fusion reaction, although it is
currently not clear whether tethering is entirely independent of the presence of the SNARE proteins.
The tethering factors so far described include the exocyst (also referred to as the sec6/8 complex in mammalian cells), which is a complex of eight proteins originally
identified in yeast as being required for the delivery of
Golgi-derived vesicles to the plasma membrane, and
which has also been found in mammalian cells to be
associated with sites of polarized secretion (Hsu et al.,
1996; Kee et al., 1997; TerBush et al., 1996). Likewise,
the large TRAPP I complex is required for ER to Golgi
transport and is localized on the early Golgi, and the
Vps52/53/54 complex has been suggested to be required for tethering of endosome-derived vesicles to the
late Golgi (Conibear and Stevens, 2000; Sacher et al.,
2001). In addition, a number of transport steps require
long coiled-coil proteins such as p115/Uso1p for membrane fusion events in the Golgi, and EEA1 for endosome
fusion, and these have also been found to contribute to
the tethering of vesicles or organelles (Cao et al., 1998;
Christoforidis et al., 1999; Waters and Hughson, 2000).
Precisely how these different factors contribute to a
given transport step and what interactions are key to
the provision of specificity is still to be elucidated. In
several cases, the tethering factors have been shown
to be recruited by rab proteins, members of the large
family of small GTPases found associated with specific
organelles of the exocytic and endocytic pathways (Zerial and McBride, 2001). Indeed, the apparent lack of
homology between different tethering complexes found
in different parts of the cell, in contrast to the families
of related SNARE proteins and rab proteins, raises the
possibility that the complexes may not serve precisely
the same role in each case.
Another protein complex that has been proposed to
have a role in vesicle tethering is the Sec34/35 complex.
The yeast genes SEC34 and SEC35 were originally identified as being required for transport through the early
part of the secretory pathway (Wuestehube et al., 1996).
Characterization of the corresponding proteins revealed
them both to be components of a large complex that is
localized to the Golgi in yeast and mammalian cells (Kim
et al., 1999; Suvorova et al., 2001; VanRheenen et al.,
1999). The complex was originally proposed to be involved in ER to Golgi transport, but recent evidence has
suggested that it may be involved in retrograde vesicle
transport in the Golgi (Spelbrink and Nothwehr, 1999;
Suvorova et al., 2001). The large size (ⵑ500–700 kDa)
and presence of additional proteins in immunoprecipitates indicated that the Sec34/35 complex contained
several further components (Kim et al., 1999; Van
Rheenen et al., 1999). This paper reports the characterization of an unexamined yeast gene that we identified
as being required for growth in cells lacking the exchange factor for Ypt6p, the yeast homolog of rab6.
Characterization of this protein, which we term Dor1p,
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528
revealed that it was associated with Sec34p and
Sec35p, and we were able to identify five further uncharacterized proteins that were also associated with this
complex. The phenotypes of yeast lacking four of these
genes are distinct from those lacking Sec34p and
Sec35p, suggesting that the complex may be involved
in multiple transport steps in the Golgi. Moreover, components of the Sec34/35 complex were found to be
related to those from the exocyst and the Vps52/53/
54 complex, suggesting that all three complexes may
perform analogous roles in different membrane traffic
steps.
Results
Identification of Dor1p
Rab6 is well conserved in evolution, but the yeast homolog Ypt6p is nonessential and shows only a slight vacuolar sorting defect when deleted (Li and Warner, 1996;
Tsukada and Gallwitz, 1996). However, it has been found
that a number of other nonessential genes become essential in the absence of YPT6. One such gene is IMH1,
the only yeast member of a family of coiled-coil proteins
that are characterized by a GRIP domain and in mammalian cells are found on the trans-Golgi (Li and Warner,
1996; Munro and Nichols, 1999; Tsukada et al., 1999).
This raised the question of whether there were further
Golgi proteins that would become essential in the absence of Ypt6p activity. To pursue this, we performed
a synthetic lethal screen using RIC1, a gene that encodes a subunit of the Ypt6p GTP/GDP exchange factor
(GEF) (Siniossoglou et al., 2000). RIC1, like YPT6, is
nonessential, and mutagenized yeast were screened for
the inability to lose RIC1 using a sectoring assay. Nineteen mutants were identified, and the corresponding
genes cloned from a yeast genomic library based on
their ability to restore sectoring. Three mutants were
complemented by IMH1, as expected from its known
synthetic lethality with YPT6 and RIC1. Of the other
mutants, one was found to be complemented by a previously uncharacterized open reading frame (ORF),
YML071c, and was selected for further analysis. Deletion of YML071c results in normal growth, but as expected cells also carrying a deletion of RIC1 were unable
to lose a plasmid-borne copy of RIC1, and so we shall
refer to YML071c as DOR1 (dependent on RIC1).
Dor1p Is Related to a Component of the Exocyst
DOR1 is predicted to encode a soluble protein of 607
amino acids, and database searches revealed a single
well-conserved homolog in the genomes of human, C.
elegans, D. melanogaster, and A. thaliana (Figure 1).
More strikingly, searches with the members of the Dor1p
family using the iterative search program PSI-BLAST
(Altschul et al., 1997) revealed a significant homology to
Sec5, a component of the exocyst (or sec6/8 complex), a
complex of eight proteins involved in the fusion of vesicles at the plasma membrane in both yeast and mammalian cells (Kee et al., 1997; TerBush et al., 1996). Homology was also found to an uncharacterized family of
proteins with a single member in each eukaryote so far
examined. This family is represented in humans by a
predicted gene called c11orf2 or ANG2 (another new
gene) (Lemmens et al., 1998; O’Brien et al., 2000). These
homologies could be found by searching with members
of any of these three families. Thus, an iterative search
with human Dor1p finds the ANG2 family and then the
Sec5 family, while a search with Sec5 family members
finds the ANG2 family and then the Dor1p family. Indeed,
a simple BLAST search with ANG2 reveals a significant
homology to members of both the Sec5p and Dor1p
families (the closest of each being Arabidopsis Sec5
[E ⫽ 9 ⫻ 10⫺9] and Drosophila Dor1 [E ⫽ 4 ⫻ 10⫺7]).
Taken together these searches indicate that Dor1p is the
yeast homolog of a protein present in higher eukaryotes,
and this protein is related to two further proteins, one
of which is a component of the exocyst. This raised
the possibility that Dor1p could be a component of an
exocyst-like complex that functions in a Golgi membrane trafficking step that becomes essential in the absence of Ypt6p activity.
Dor1p Is Associated with Seven Other Proteins
To investigate the possibility that Dor1p is part of a
larger complex, we made use of the protein A tagging
system that has been used to isolate a number of protein
complexes from yeast (Grandi et al., 1993; Jungmann
et al., 1999). Homologous recombination was used to
insert two copies of the IgG binding Z domain from
protein A at the C terminus of the DOR1 gene in a protease-deficient yeast strain. Lysates were prepared in parallel from the tagged strain and from a control strain,
and passed over IgG-Sepharose. The bound proteins
were eluted and analyzed by gel electrophoresis. Figure
2 shows that several proteins were specifically isolated
from the tagged strain, including a band of the expected
size for protein A-tagged Dor1p (Dor1p-ZZ). The coprecipitating proteins were identified by subjecting the
bands to in-gel tryptic digestion, and determining the
masses of the tryptic fragments by mass spectrometry.
This revealed the presence of Dor1p-ZZ as expected,
but also seven further proteins that had coprecipitated
with Dor1p (Figure 2). These included five previously
uncharacterized proteins, and the proteins Sec34p and
Sec35p encoded by genes identified as being required
for transport through the early parts of the secretory
pathway (Wuestehube et al., 1996).
To confirm the mass spectrometric identification of
the proteins, yeast strains were constructed in which a
protein A tag was attached to the C terminus of each
of the ORFs, and these strains then transformed with a
plasmid expressing Dor1p with a C-terminal HA tag.
The HA-tagged Dor1p was found to be present in IgGSepharose precipitations from every strain with the exception of the control strain and that expressing Dor1pZZ itself (Figure 3A). This confirmed that the proteins
identified by mass spectrometry are indeed associated
with Dor1p and indicates that Dor1p is present at only
a single copy in the complex.
Dor1p Is a Component of the Sec34/35 Complex
Two of the proteins found to be associated with Dor1p
were Sec34p and Sec35p. Previous analysis of Sec34p
and Sec35p has shown that they are components of a
large complex termed the “Sec34/35 complex” (Kim et
al., 1999; VanRheenen et al., 1999). All of the Sec34p
Characterization of the Sec34/35 Complex
529
Figure 1. The Yeast Gene DOR1 Encodes a Protein Related to Sec5, a Component of the Exocyst
BLAST searches of the GenBank database with the protein sequence encoded by the yeast gene DOR1 (YML071c) identified a single close
relative from human (Hs), C. elegans (Ce), D. melanogaster (Dm), and A. thaliana (At). An iterative PSI-BLAST search with human Dor1p
revealed homology to a human protein of unknown function and its relatives in other species [ANG2 (c11orf2), E ⫽ 6 ⫻ 10⫺9 after one iteration],
and then to rat Sec5 (E ⫽ 7 ⫻ 10⫺4 after three iterations). The homology is greatest in the N-terminal half of the proteins, and these regions
are shown aligned using CLUSTAL W (Thompson et al., 1994), residues shaded if identical (black) or conserved (gray) in at least three proteins.
For the Dor1 and ANG2 families the species ORF names for each gene are stated. The sequence of human Dor1 was assembled from
overlapping ESTs identified by homology to murine Dor1 (GenBank accession no. BAA95093).
and Sec35p in cells appears to fractionate in a single
peak, indicating that there is no substantial pool of protein outside this complex. To investigate whether the
five Dor1p-associated proteins identified here are all
associated with the Sec34/35 complex, as well as with
Dor1p, the IgG-Sepharose precipitates of the protein
A-tagged components were probed with an antibody
against Sec35p. Figure 3B shows that Sec35p coprecipitated with each of these proteins, and a similar result
was also obtained with antibodies to Sec34p (data not
shown). The most parsimonious interpretation of these
results is that Dor1p, and the five further novel proteins,
are all components of the Sec34/35 complex. It remains
formally possible that there are in fact two classes of
complex, both of which contain Dor1p, Sec34p, and
Sec35p, although previous analysis of Sec34p and
Sec35p has suggested that they behave as components
of a single complex. Moreover, when Dor1p-ZZ was
eluted from beads by specific protease cleavage of the
linker between the protein and the tag, and the released
complexes examined by negative stain electron microscopy, a relatively homogeneous population of particles
was observed (Figure 3C). These particles were 250 ⫻
290 angstroms in their largest projection, and 210 ⫻ 250
angstroms in their smallest projection, with a similar
appearance when observed unstained in vitreous ice (P.
Rosenthal, personal communication). Taken together,
these results indicate that Dor1p is associated with
Sec34p, Sec35p, and five further proteins, and together
these eight proteins comprise the Sec34/35 complex.
Properties of the Dor1p-Associated Proteins
Five of the proteins identified above have not been previously characterized, apart from the corresponding
genes having been deleted in the global analysis of the
yeast genome. We will refer to these genes as COD
genes, [complexed with Dor1p; YPR105c (COD1),
YNL041c (COD2), YGL223c (COD3), YNL051w (COD4),
YGL005c (COD5)]. Database searches revealed that
three have closely related homologs in higher eukaryotes (COD1, COD2, and COD4), and the known properties of these yeast genes, and the homologs in other
species, are given in Table 1.
Examination of the properties of these new components of the Sec34/35 complex revealed several notable
features. First, like Dor1p, for those Cod proteins that
are conserved in other species, a single homolog can
be found in each eukaryote with a sequenced genome,
suggesting that these proteins will be found in all eukaryotes. Second, Cod4p is the yeast homolog of mammalian GTC-90, the only identified component of the “Golgi
transport complex” (GTC) that was purified from bovine
brain as being required for intra-Golgi transport in vitro
(Walter et al., 1998). The function of this complex is
unknown, but it comprises at least five proteins. The
third feature of the seven yeast proteins associated with
Dor1p is that they do not all show identical phenotypes
when deleted. DOR1, COD2, COD4, and COD5 are all
nonessential genes, and null strains showed growth
rates indistinguishable from wild-type (data not shown).
In contrast, COD1 has been reported to be essential
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530
higher and lower temperatures (Kim et al., 1999; Spelbrink and Nothwehr, 1999; VanRheenen et al., 1998).
Likewise, strains deleted for COD3 showed greatly
slowed growth at 30⬚C and near inviability at 37⬚C (data
not shown). Thus, four of the components of the Sec34/
35 complex show severe growth defects when deleted,
and four do not seem to be required for normal growth.
Figure 2. Dor1p Is Associated with Seven Other Proteins
Eluates from IgG-Sepharose beads incubated with lysates from 40
g of yeast expressing a protein A tagged form of Dor1p or from the
same mass of a control strain (–). Eluted proteins were separated by
gel electrophoresis and stained with Coomassie blue. The indicated
bands were identified by mass spectrometry of tryptic fragments.
Sec34p was mainly present as two bands smaller than its predicted
molecular weight, but this is apparently due to postlysis proteolysis
as a full-length version of the protein, which comigrates with Cod1p
and Cod2p, was found in smaller scale precipitations performed in
the absence of detergent. Two of the minor bands corresponded to
Cod1p and Cod2p (*) and presumably represent proteolysis during
isolation. Ssa1/2p are hsp70s, and frequent contaminants of precipitations, with a comigrating band also being present in the control
lane.
when deleted, and sec34 and sec35 were originally isolated as temperature sensitive mutants that were inviable at 37⬚C (Wuestehube et al., 1996). Subsequently, it
was reported that deletion of either gene resulted in a
very severe growth defect at 30⬚C, with lethality at both
Analysis of the Phenotypes of Mutations
in Dor1p-Associated Proteins
In order to understand more about the function of Dor1p
and its associated proteins, we examined in more detail
the strains lacking the four nonessential components,
as well as the slow-growing strain lacking COD3. When
examined by electron microscopy, the internal membrane compartments appeared grossly normal in the
four strains lacking either DOR1, COD2, COD4, or COD5
(Figure 4 and data not shown). In contrast, the strain
lacking COD3 showed an abnormal accumulation of internal membranes that appeared at least similar to that
seen with the temperature sensitive strain sec35-1 at
the nonpermissive temperature.
Mutants of SEC34 have previously been found to have
defects in the elaboration of N-glycans that occurs in
the yeast Golgi (Spelbrink and Nothwehr, 1999; Wuestehube et al., 1996). The gel mobility of the heavily glycosylated secreted protein invertase is sensitive to defects
in Golgi glycosylation, and Figure 5A shows that while
invertase mobility is at most only slightly affected in the
four nonessential deletion strains, it is increased in the
⌬cod3 strain, indicating that Golgi glycosylation is
greatly reduced.
We also examined the recycling of proteins back to
the Golgi from the endocytic pathway. The v-SNARE
Snc1p acts in the fusion of Golgi-derived vesicles with
the plasma membrane, and it then recycles back to the
Golgi via the endocytic pathway (Lewis et al., 2000). The
distribution of GFP-Snc1p was thus examined in the
deletion strains. In wild-type cells, the protein was localized primarily to the plasma membrane as expected.
Figure 3. Protein A-Tagged Cod1–5p Are Associated with Dor1p and Sec35p
(A) Protein blots of total cellular protein, or of
IgG-Sepharose precipitates, from yeast strains
in which the only copy of the indicated gene
is tagged with protein A, or no gene is tagged
(control). The strains also contain a plasmid
expressing HA-tagged Dor1p.
(B) Protein blots of the IgG-Sepharose precipitates from the same strains as in (A), but
probed with rabbit anti-Sec35p, which also
binds to the protein A fusions (*).
(C) Electron micrograph of negatively stained
Dor1p-containing particles. IgG-Sepharose
precipitates were prepared as in A, but the
protein eluted in native conditions with TEV
protease, applied to grids and stained with
2% uranyl acetate for microscopy.
Characterization of the Sec34/35 Complex
531
Table 1. Properties of Dor1p, Its Associated Proteins, and Their Homologs
Gene
ORF Name
Size (aa)
Phenotype
of Deletion
DOR1
YML071c
607
viable
2
COD1
YPR105c
861
inviable
COD2
YNL041c
839
viable
COD3
COD4
YGL223c
YNL051w
417
403
very slow growth ts
viable
COD5
SEC34
YGL005c
YER157w
279
801
viable
very slow growth ts
SEC35
YGR120c
275
very slow growth ts
Homologs in Higher Eukaryotes
(size in aa)
Human
Drosophila
C. elegans
Arabidopsis
FLJ22315
(613)1
AK0202874
(785)
AF116827
(653)3
—
GTC-904
(839)
—
hSec345
(828)
—6
CG6488
(570)
CG7456
(761)
CG1968
(630)
—
CG6549
(751)
—
CG3248
(883)
R02D3.2
(743)
Y51H7C.6
(801)
K07C11.9
(642)
—
C43E11.11
(802)
—
Y71F9AM.6
(670)
F14F18.150
(619)
F3D13.1
(991)
F5M6.21
(706)
—
T23K23.22
(832)
—
TPL24.37
(745)
1
FLJ22315 is a partial cDNA clone, full-length protein assembled from ESTs (see Experimental Procedures).
Inviable according to a high throughput screen of the yeast genome (Winzeler et al., 1999) but is in fact able to grow very slowly (see
Discussion).
3
Full-length protein assembled by reference to a second overlapping cDNA (GenBank no. BAA86448).
4
Walter et al. (1998)
5
Suvorova et al. (2001)
6
Sec35p has no close homologs in higher eukaryotes, but by PSI-BLAST the ldlCp family is more closely related to Sec35p than to any other
protein in S. cerevisiae, and so it is possible that the proteins are distantly related orthologs.
2
However, in the ⌬dor1 strain, GFP-Snc1p accumulated
in internal membranes, and this phenotype was also
seen in the strains lacking COD2, COD4, or COD5. The
slow-growing ⌬cod3 strain also showed internal accumulation of GFP-Snc1p, but the distribution is more diffuse, perhaps reflecting the greater perturbation of internal membranes in these cells. Thus, deletion of any one
of the four genes DOR1, COD2, COD4, or COD5 causes
no substantial defect in growth, internal membrane organization, or Golgi processing of invertase, processes
that are all affected in strains lacking Cod3p, Sec34p,
and Sec35p. However, these four genes do have defects
in the recycling of the v-SNARE Snc1p. Taken together,
these results suggest that the Sec34/35 complex may
contribute to more than one membrane traffic step in
the Golgi.
Human Homologs of Dor1p and Cod1p Are
Localized to the Golgi
Of the six new components of the yeast Sec34/35 complex that we report in this paper, four have closely related homologs in higher eukaryotes. As mentioned
above, Cod4p is homologous to mammalian GTC-90, a
component of the GTC that is present on Golgi membranes (Walter et al., 1998). To examine the significance
of the other homologies, full-length human cDNA clones
encoding homologs of Dor1p and Cod1p were epitope
tagged at the C terminus and expressed in COS cells.
At low expression levels, both proteins showed clear
localization to the Golgi apparatus (Figure 6). This localization tended to be around the edges of the Golgi,
a distribution previously seen with antibodies to COPI
components and to giantin, proteins localized to Golgi
rims rather than within the cisternae (Shima et al., 1997).
At higher levels of expression, a diffuse cytosolic staining was observed, presumably reflecting saturation of
available binding sites on the Golgi or in the complex,
and a concomitant accumulation of the unassembled
protein in the cytosol where it is apparently stable.
Relationship between the Sec34/35 Complex
and Other Transport Complexes
As discussed above, Dor1p is distantly related to the
Sec5 component of the exocyst. We thus investigated
the possibility that other components of the Sec34/35
complex might also be related to exocyst components.
One feature of the eight proteins that comprise the exocyst is that near their N termini are short regions predicted to be capable of forming coiled-coils (Kee et al.,
1997; TerBush et al., 1996). Examining the eight yeast
components of the Sec34/35 complex with the COILS
prediction program (Lupas et al., 1991) revealed the
same was also true for these proteins and also for their
homologs in other species. More strikingly, iterative PSIBLAST searches with the N-terminal regions of several
of the Sec34/35 and exocyst components consistently
found statistically significant matches (E ⬍ 0.005) to the
similar regions of other such components, ahead of any
other coiled-coil proteins. These regions, aligned in Figure 7A, are most clearly related over a region predicted
to form two amphipathic helices separated by an extended loop. Interestingly, these searches also found
significant homology to the N-terminal regions of ldlBp
and ldlCp, proteins found in a complex of unknown function on the Golgi in mammalian cells, which has been
suggested to possibly correspond to the GTC that contains the human homolog of Cod4p (Chatterton et al.,
1999). Moreover, homology was also observed to the
N-terminal regions of the proteins Vps53p and Vps54p,
components of a recently identified trimeric complex
proposed to be involved in tethering endosome-derived
vesicles to the late-Golgi (Conibear and Stevens, 2000).
For some of these transport complex components, the
homology revealed by PSI-BLAST searches extended
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532
beyond the N-terminal amphipathic helices, suggesting
a closer relationship than simply a shared domain. In
particular, searches with either Exo70 from the exocyst,
or Sec34, showed that they have extended homology
to each other and, more distantly, to the Cod2 family
(Figure 7B). In addition, the exocyst component Sec3
and Vps52 also show an extended region of homology
to each other (Figure 7C). Thus, it appears that all three
of these complexes are structurally related, suggesting
they may perform closely related functions.
Discussion
Figure 4. Deletion of COD3 Results in Aberrant Accumulation of
Internal Membranes
Electron micrographs of sections of the indicated yeast strains following permanganate fixation and embedding (Kaiser and Schekman, 1990). The ⌬cod3 strain contains many small darkly staining
structures and has fragmented vacuoles. This resembles sec35-1
cells fixed an hour after being elevated to the nonpermissive temperature. In contrast, ⌬dor1 and ⌬cod2 appear similar to wild-type
(BY4741), and a similar result was obtained for ⌬cod4 and ⌬cod5
(data not shown). Scale bar, 2 ␮m.
In this paper, we describe a yeast protein, Dor1p, which
we identified as being essential in cells that lack the
Ypt6p exchange factor Ric1p. Identification of the seven
proteins found associated with Dor1p revealed it to be
a component of the Sec34/35 complex. We were able
to identify all of the more abundant proteins that we
found coprecipitating with Dor1p, and all of the fainter
bands examined were found to be either breakdown
fragments of these proteins or abundant cytosolic proteins, such as heat shock proteins that we have frequently found in isolates of protein complexes and presumably bind nonspecifically. Thus, it appears that the
complex comprises eight proteins, although we cannot
exclude the possibility that further more minor, or loosely
associated, components exist. Two previous gel filtration analyses of the Sec34/35 complex indicated sizes
of ⵑ480 kDa or up to 750 kDa (Kim et al., 1999; Van
Rheenen et al., 1999). If each of these eight proteins is
present as a single copy, then the total predicted mass
would be 515 kDa, which at least lies within this range.
Analysis of the six proteins associated with Sec34/35
revealed that like Sec34p itself, four (Dor1p, Cod1p,
Cod2p, and Cod4p) have clear homologs in higher eukaryotes. Of these homologs the only known one is GTC90, a component of GTC, a multiprotein complex purified
as an activity in cytosol that stimulated an in vitro Golgi
transport assay (Walter et al., 1998). No other components of this complex have been reported, but it seems
quite possible that the GTC is the mammalian version
of the Sec34/35 complex.
Figure 5. Membrane Trafficking Phenotypes of Strains Lacking DOR1 and COD1–5
(A) Anti-myc protein blots of total cellular protein from yeast strains expressing myc-tagged version of the secreted glycoprotein invertase.
⌬mnn9 mutants lack all of the mannan structure that is attached in the Golgi to the N-glycans of invertase and other glycoproteins.
(B) Confocal micrographs of live cells of the indicated yeast strains expressing a GFP-tagged version of the v-SNARE Snc1p. The fusion is
expressed from a CEN plasmid that gives no greater than twice the level of the endogenous protein (Lewis et al., 2000). Scale bar, 2 ␮m.
Characterization of the Sec34/35 Complex
533
Figure 6. Human Homologs of Dor1p and Cod1p Are Found on the
Golgi
Confocal micrographs of COS cells transfected with plasmids encoding triple myc-tagged cDNAs of human Dor1 or Cod1. Cells were
fixed, permeabilized, and stained with antibodies against the myc
tag and the indicated Golgi marker. At higher expression levels, the
tagged proteins accumulated throughout the cytoplasm. Scale bars,
5 ␮m.
What can be said about the function of the Sec34/35
complex? The original sec34-1, sec34-2, and sec35-1
mutants were temperature sensitive for growth, and at
the nonpermissive temperature showed reduced secretion of invertase, and accumulated forms of carboxypeptidase Y (CPY) that lacked Golgi modifications
(Wuestehube et al., 1996). Analysis of ER to Golgi transport in semipermeabilized cells indicated that at the
nonpermissive temperature, budding of ER-derived vesicles was normal, but their subsequent tethering to the
Golgi was reduced, leading to the suggestion that the
Sec34/35 complex could be required to tether ERderived vesicles to the early Golgi (VanRheenen et al.,
1998). However, subsequent work showed that unlike
most other genes involved in ER to Golgi transport,
neither SEC34 nor SEC35 is absolutely essential for
growth, although ⌬sec34 and ⌬sec35 cells grow very
slowly and their secreted proteins have defects in Golgi
glycosylation (Kim et al., 1999; Spelbrink and Nothwehr,
1999; VanRheenen et al., 1999). Indeed, the TRAPP I
complex, most of whose subunits are essential, has
been recently found to bind ER-derived vesicles and
has been proposed to serve in tethering these vesicles
to the Golgi (Sacher et al., 2001). Moreover, the SEC34
gene was independently isolated as GRD20, a gene required for recycling of a reporter protein from endosomes back to the late Golgi (Spelbrink and Nothwehr,
1999). Finally, a human Sec34p homolog was found to be
on the Golgi itself, and not the vesicular-tubular clusters
that form in the periphery of the cell from the fusion of
ER-derived transport vesicles (Suvorova et al., 2001).
Taken together, these results suggest that the effects of
mutations in SEC34 and SEC35 on ER to Golgi transport
could be indirect consequences of defects in vesicle
recycling events in the Golgi itself.
The analysis reported here of the further six components of the Sec34/35 complex may provide a means
of reconciling the range of phenotypes associated with
sec34 mutations. Strikingly, four of the Sec34/35-associated proteins (Dor1p, Cod2p, Cod4p, and Cod5p) show
no growth defect when deleted and show no severe
defect in Golgi glycosylation. In contrast, COD3 is more
like SEC34 and SEC35 in that its deletion causes very
slow growth and glycosylation defects. COD1 was found
by the EUROFAN project to be an essential gene, but
in the light of our results we have recently reinvestigated
this, and found that spores lacking the gene do grow,
albeit very slowly (J.R.C.W. and S.M., unpublished data).
Taken together, this suggests that the Sec34p/Sec35p/
Cod1p/Cod3p components of the complex are required
for a transport step necessary for normal growth and
correct glycosylation. A clear candidate for this would
be the recycling of vesicles containing Golgi enzymes
back to the early Golgi. Indeed, the in vitro Golgi transport assay that was used to isolate the GTC from mammalian cells has been proposed to be measuring this
process (Lin et al., 1999; Pelham and Rothman, 2000).
In this model, only four of the components would be
required for this transport step, implying that the complex is also involved in a second step, which requires
the other four components. The aberrant distribution of
GFP-Snc1p in these strains suggests that this could be
recycling of vesicles from endosomes to the Golgi, but
whether to the early or late Golgi is unclear. Dor1p,
although not essential in normal cells, becomes essential when cells lack Ric1p, the GEF for Ypt6p. Ric1p is
localized to the late Golgi, and loss of the gene also
interferes with recycling of Snc1p back to the late Golgi
(Siniossoglou et al., 2000). Thus, Ypt6p and the Sec34/
35 complex could act together in a single transport step
that can function at a reduced rate in the absence of
either one. Alternatively, it has been proposed that there
are multiple routes for recycling of membranes from the
endocytic system to the Golgi (Holthuis et al., 1998;
Lupashin et al., 1997; von Mollard et al., 1997), and so
Ypt6p and the Sec34/35 complex could be involved in
tethering distinct classes of vesicle, one of which must
be correctly targeted for cells to recycle sufficient endosomal material for continued growth. The more essential
quartet of proteins could also be required for this step,
and, indeed, overexpression of the v-SNARE Snc2p has
been reported to partially suppress the temperaturesensitive phenotype of sec35-1 (VanRheenen et al.,
1998).
Finally, perhaps the most surprising aspect of the
Sec34/35 complex is the sequence homology between
Developmental Cell
534
Figure 7. Homology between Components of the Sec34/35 Complex and Other Transport Complexes
(A) Alignment of the N-terminal amphipathic helical regions of the indicated proteins: Dor1, Cod4, and Sec35p from the Sec34/35 complex,
Sec5, Sec8, and Exo84 from the exocyst, Vps53 and Vps54, the putative GTC components ldlCp and ldlBp, and a protein of unknown function,
ANG2. In each case, the human homolog is shown, with the exception of yeast Sec35p, which does not have clear homologs in higher
eukaryotes. Homology was identified by PSI-BLAST searches with full length human Dor1, Sec5, or ldlCp. In each case, significant homology
(E ⬍ 0.005) was found to the other two families, plus those of GTC-90 (Cod4), ldlBp, and ANG2 (using 5, 7, and 5 iterations for Dor1, Sec5,
and ldlCp respectively). Vps53 and Vps54 were found in addition when the search was with the first 200 residues of ldlCp. The homologous
regions were aligned in CLUSTAL W, and residues shaded if identical (black) or conserved (gray) in at least three proteins. Gray bars show
the regions predicted to form coiled-coil (the hydrophobic heptad repeat indicated by black circles), with the region between predicted by
JPred (Cuff et al., 1998) to be nonhelical, or even extended, suggesting it could form a loop.
(B) Alignment of the N-terminal portions of Sec34, Cod2, and Exo70 family members from the species indicated as in Figure 1. A PSI-BLAST
search with C. elegans Exo70 (C43E11.8) showed highly significant homology to Sec34 and Cod2 family members after three iterations. The
gene names for the Sec34 and Cod2 families are in Table 1. For Exo70, Dm is CG7127 and At is F12E4.320. Alignment and shading as in (A).
(C) Alignment of portions of Sec3 and Vps52 family members from the indicated species. A PSI-BLAST search with full length Drosophila
Sec3 finds a member of the Vps52 family after a single iteration (E ⬍ 0.001), and the rest after a further iteration (E ⫽ 2 ⫻ 10⫺27 for human
Vps52). Species indicated as Figure 1, genes for Sec3 being Ce, F12E4.7 and At, F16N3.18; and for Vps52, Dm, CG7371; Ce, F08C6.3; and
At, F3I17.5.
Characterization of the Sec34/35 Complex
535
its components and those of the exocyst and the Vps52/
53/54 complex. These relationships were not noted
when the proteins were originally reported, but the recent completion of several eukaryotic genomes has
greatly increased the number of orthologs for any given
protein. A diverse set of orthologs is required for iterative
search programs such as PSI-BLAST to be able to identify the residues conserved within a family, and so find
distant relationships to other protein families (Altschul et
al., 1997). The homology between the different tethering
complex components is mostly restricted to a region
near the N termini of proteins that is predicted to form
short amphipathic helixes, or coiled-coils. However, for
some sets of proteins, such as Dor1p and Sec5p, or
Sec34p and Exo70p, the homology extends further, suggesting they may share structural or functional features
not found in other components. It has previously been
suggested that the putative coiled-coil regions at the
end of the exocyst components could be involved in
holding the complex together (Kee et al., 1997; TerBush
et al., 1996). However, yeast Sec3p has been found to
bind directly to Sec5p, and this interaction does not
require the putative coiled-coil region of Sec3p (Guo et
al., 2001). Moreover, in mammalian Exo84 the amphipathic helixes are separated from the rest of the protein
by a PH domain, perhaps more consistent with an exposed location near the membrane. Thus, it remains
possible that this structure has a functional role in binding other proteins involved in vesicle docking and fusion,
possibly though coiled-coil interactions. Interestingly,
the mammalian exocyst has been reported to coprecipitate with the plasma membrane t-SNARE syntaxin (Hsu
et al., 1996).
The Vps52/53/54 complex has only recently been
identified, and little is known about how it functions
beyond the fact that it is localized to the late Golgi and
is required for the fusion of endosome-derived vesicles
(Conibear and Stevens, 2000). However, more is known
about the function of the exocyst. Like the Sec34/35
complex, it has eight components, and in yeast loss of
function of any one results in a block in fusion of Golgiderived vesicles to the plasma membrane. The precise
role of the complex in this process is not certain, but it
is recruited to vesicles by the small GTPase Sec4p and
to sites of polarized cell growth by Rho3p, clearly suggesting that it may serve to connect the vesicle to the
intended site of delivery (Guo et al., 1999, 2001). Whether
the complex also plays a role in the subsequent assembly of SNARE complexes and membrane fusion is not
at present clear. The exocyst in mammals (where it is
also referred to as the sec6/8 complex) is apparently
expressed in all tissues but is often found to be strikingly
concentrated at particular sites on the plasma membrane (Hsu et al., 1999). Thus, the complex is localized
in axon growth cones, in forming synapses, and at sites
of cell-cell contact in polarized epithelial cells (Grindstaff
et al., 1998; Hazuka et al., 1999). However, the complex
is absent from mature synapses, and antibodies against
mammalian Sec6 block delivery to the basolateral, but
not apical surface, of permeabilized epithelial cells. This
raises the possibility that the complex is not obligatory
for all vesicle fusion events with the plasma membrane,
but rather can serve to ensure that fusion is restricted
to particular regions of the plasma membrane. Within
the Golgi, there is only a limited number of t-SNARE
proteins to mediate fusion with incoming vesicles, and,
indeed, these are able to form a wide range of possible
complexes with v-SNAREs in vitro (Pelham, 2001; Tsui
et al., 2001). This means that vesicles are likely to contain
v-SNAREs that could assemble with the t-SNAREs on
multiple cisternae. Tethering complexes could serve to
restrict fusion to a subset of these cisternae, and so
ensure accurate recycling of vesicles within the Golgi.
However, it is possible that all these complexes could
also have a more direct role in subsequent membrane
fusion events. The identification of families of components such as rabs and SNAREs that act in multiple
steps in membrane trafficking has helped to advance
understanding of the molecular processes of vesicle
transport. The relationship described here between the
Sec34/35 complex, the exocyst, and the Vps52/53/54
complex adds another set of components conserved
between different transport steps. Information obtained
from one complex may well be helpful in understanding
the function and structure of the other complexes and,
hence, the mechanisms of vesicle trafficking.
Experimental Procedures
Yeast Strains
The wild-type background for the synthetic lethal screen was IAY11
(MAT␣ ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3-52 ade3⌬853; based on K700, Nasmyth, IMP, Vienna) and for protein A tagging
was the protease-deficient strain c13-ABYS-86 (MAT␣ pra1-1 prb1-1
prc1-1 cps1-3 ura3⌬5 leu2-3 his [Heinemeyer et al., 1991]). Strains
lacking DOR1 and COD1-5 were obtained from EUROSCARF in the
background BY4741 (MATa his3⌬1 leu2⌬0 lys2⌬0 ura3⌬0 [Brachmann et al., 1998]). Except where stated, other genes were deleted
or epitope tagged using the PCR method and the kanMX marker
(Baudin et al., 1993).
Synthetic Lethal Screen
RIC1 was deleted in strain IAY11 using the ⌬ric1::HIS3 disruption
plasmid pEI, to give strain JWY88. Disruption of RIC1 was confirmed
by colony PCR and by the gain of a temperature-sensitive growth
phenotype that was complemented by reintroduction of RIC1 on
a plasmid (pEH: RIC1, ADE3, URA3). JWY88 containing pEH was
mutagenized by ultraviolet irradiation to 99% cell death, and 13,000
colonies screened visually for their inability to sector on low adenine
plates. Nonsectoring colonies were twice restreaked and examined
for sectoring and then streaked onto 5-fluoroorotic acid (FOA) plates
to confirm their inability to lose pEH. Of the 27 mutants that failed
to grow on FOA, 19 regained sectoring when transformed with RIC1
(pFV, based on TRP CEN vector pRS314), but not with the empty
vector, indicating that they are bona fide synthetic lethal mutants.
Of these, three sectored when transformed with IMH1 in a TRP
plasmid (pFW). Plasmids complementing the remaining mutants
were sought by transformation with a centromeric genomic library
in the TRP vector pRS200 (ATCC no. 77164). One of the mutants
sectored when transformed with a plasmid containing a part of
chromosome 13, and subcloning revealed the responsible ORF to
be YML071c (DOR1). A ⌬dor1::LEU2 deletion plasmid (pLX) was
used to delete DOR1 in JWY88 carrying pEH, and the deletion mutant
was unable to sector or grow on FOA. Transformation with TRP
plasmids carrying either RIC1 or DOR1 restored the ability to sector
and grow on FOA.
Protein A Tagging and Immunoprecipitation
DOR1 and other genes were tagged at their C termini in the genome
of the protease-deficient strain c13-ABYS-86 by transformation with
PCR products from the template plasmid pFZ, encoding the protein
A tag, the 3⬘ UTR from the ADH1 gene and the dominant marker
kanMX. Integration of this product results in fusion to the DOR1
ORF of a 15.6 kDa tag encoding a linker sequence (GAGAGA), a
Developmental Cell
536
cleavage site for tobacco etch virus (TEV) protease, and two copies
of the IgG binding (Z) domain of S. aureus protein A (Rayner and
Munro, 1998).
For large-scale immunoprecipitation of Dor1p-ZZ, 2 liter cultures
were grown to OD600 of about 10, harvested, and washed once in
distilled water. The pellet was resuspended to an OD600 of 50 (“one
spheroplasting volume”) in 100 mM Tris, pH 9.4, 50 mM ␤-mercaptoethanol, incubated for 10 min at room temperature, pelleted, and
the mass of the pellet determined. Cells were washed in one spheroplasting volume of ice-cold spheroplasting buffer (1.4 M sorbitol,
50 mM potassium phosphate, pH 7.5, 10 mM NaN3), resuspended
in the same volume of spheroplasting buffer prewarmed to 37⬚C,
zymolyase 100T added [0.5–1.0 mg/(g of cell pellet), ICN Biomedicals], and gently agitated at room temperature until spheroplasting
was 80%–90% complete as determined by lysis upon dilution in
water (typically 30 min). All subsequent steps were carried out at
4⬚C using cold solutions. Spheroplasts were pelleted and resuspended with vigorous vortexing in 90 ml lysis buffer (20 mM Tris,
pH 8.0, 150 mM KCl, 5 mM MgCl2) supplemented by protease inhibitors. Ten milliliter 10% (v/v) Triton X-100 was added, spheroplasts
dounce homogenized (15 strokes), and the lysate centrifuged at
15,000 rpm for 10 min in a Sorvall SS34 rotor. The supernatant
was incubated with 300 ␮l Sepharose CL-6B (Amersham Pharmacia
Biotech) for 1 hr, the Sepharose pelleted, and the supernatant incubated overnight with 300 ␮l acid-washed IgG-Sepharose 6 Fast
Flow resin (Amersham Pharmacia Biotech). Beads were then
washed once in 50 ml lysis buffer containing 1% Triton X-100, transferred to a Mobicol 1 ml column (MoBiTec), and washed three times
with 0.5 ml lysis buffer and twice with 0.5 ml 5 mM NH4OAc (pH
5.0). Proteins were eluted with 500 ␮l 0.5 M acetic acid (pH 3.4), the
eluate lyophilized, boiled in 1⫻ SDS sample buffer and loaded on
a 6%–15% SDS polyacrylamide gradient gel. Bands were excised,
digested with trypsin, and subjected to matrix-assisted laser desorption ionization mass spectrometry (Shevchenko et al., 1996).
For negative staining, preparation was as above except that Triton
X-100 was omitted, and instead of using acetic acid, the complex
was eluted by adding 0.5 bead volumes of lysis buffer containing
200 units/ml TEV protease (Life Technologies), incubating at room
temperature for 10 hr and collecting the eluate by brief centrifugation. Small scale precipitations from 100 ml cultures for Western
blotting were as above, except Triton X-100 was omitted, and acid
used for the elution, with all other volumes reduced accordingly.
Protein Blots
Myc-tagged invertase (Jungmann et al., 1999) was expressed from
a constitutive PHO5 promoter in a CEN URA plasmid (pUinvmyc),
and triple HA-tagged Dor1p from its own promoter in CEN plasmid
pLM (based on pRS316). Proteins transferred on to nitrocellulose
were detected with antibodies to the Myc epitope tag (rabbit antimyc, Santa Cruz), the HA epitope (12CA5), or Sec35p (VanRheenen
et al., 1998), and peroxidase conjugated species-specific antibodies, followed by chemiluminescence (ECL, Amersham Pharmacia
Biotech). Protein A tags were detected with peroxidase rabbit antiperoxidase (DAKO).
Mammalian Cells
ESTs from the IMAGE consortium were used to assemble full-length
cDNA clones for human Dor1 (GenBank accession nos. AI858625
and BE885876) and Cod1 (AI634312). A number of the Dor1 ESTs
(including AI858625) predict a shorter version of the protein, but
this represents translation into an intron which is spliced out in
the longer versions to attach further protein-encoding exons. The
significance of these shorter versions is unclear, and for these studies the longer version of the protein encoded by BE885876 was
used. The cDNAs were cloned into a COS cell expression vector
containing the CMV promoter, with a short linker and three copies
of the Myc epitope tag [gtgagaga(eqkliseedlg)2eqkliseedlag]
attached to the end of the ORFs so that the resulting proteins were
MATAATIP…VGPgtga… (Dor1p) and MGTKMADLD…KRLRLgtga…
(Cod1p), the native proteins in upper case. COS cells were transfected with Fugene (Roche), split on to glass slides and 24–30 hr
after transfection fixed with 4% paraformaldehyde. Cells were permeabilized with 0.5% Triton X-100 in PBS, blocked with 20% fetal
calf serum/0.25% Tween 20/ PBS, and stained with antibodies in
the same solution. Rabbit anti-Myc (Santa Cruz); rat anti ␤⬘COP
(23C; Harrison-Lavoie et al., 1993); and mouse anti-p115 and antiGolgin-245 (p230) (BD Transduction Labs) were detected with appropriate Alexa-labeled secondary antibodies (Molecular Probes),
and fluorescent images obtained on an MRC600 confocal microscope (Bio-Rad).
Acknowledgments
We are indebted to Sew-Yeu Peak-Chew and Farida Begum for
protein identification by mass spectrometry, and grateful to Rainer
Duden, Peter Rosenthal, Symeon Siniossoglou, and Gerry Waters
for advice and reagents, and to Alison Gillingham and Katja Röper
for comments on the manuscript.
Received August 24, 2001; revised September 14, 2001.
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