Much effort has been extended towards gaining an understanding of the mechanism of transport vesicle docking in the secretory pathway. Several families of proteins are involved in this event, including the rab family of small GTP-binding proteins and the SNARE family of integral membrane proteins. Recently, another class of proteins has been described, the tethering factors. Although these proteins do not display homology with one another and thus do not define a family, they share a similar function in docking, that of connecting the vesicle to the target compartment before the interaction of v- and t-SNAREs (for reviews see Pfeffer 1999
; Waters and Pfeffer 1999
). The docking event can therefore be separated into two distinct substages, tethering and SNARE-dependent docking.
A recent genetic screen identified temperature-sensitive alleles of two genes, SEC34
, that, when incubated at the restrictive temperature, are defective in ER to Golgi complex transport and accumulate large numbers of vesicles ( Wuestehube et al. 1996
). Mutant alleles of these genes are also able to be suppressed by the dominant allele of SLY1
, a trait shared with all previously characterized ER to Golgi complex tethering factors ( Sapperstein et al. 1996
; Cao et al. 1998
; VanRheenen et al. 1998
). Based on these data, we hypothesized that SEC34
might be involved in tethering, and this was demonstrated to be the case for SEC35
by the discovery that this gene both displayed a genetic interaction with genes involved in tethering and is required in this process as revealed by an in vitro assay ( VanRheenen et al. 1998
). We therefore investigated whether Sec34p functions in tethering as well.
To begin our study of SEC34, we cloned the gene by complementation of the temperature-sensitive phenotype of a strain bearing the sec34-2 mutation. SEC34 was discovered to be a novel gene encoding a protein with a predicted molecular weight of 93 kD. Deletion of SEC34 in a haploid strain resulted in a severe growth defect, and thus SEC34 is essential for wild-type growth rates, although not for viability.
To investigate the genetic interactions of SEC34 we employed multicopy suppressor analysis. The best suppression of the sec34-2 temperature-sensitive growth defect was conferred by overexpression of Ypt1p, the rab required in ER to Golgi complex transport, or by expression of Sly1-20p, the dominant form of the t-SNARE–associated factor, Sly1p. Suppression of the SEC34 deletion strain allowed us to order the action of Sec34p with respect to Ypt1p and Sly1p. Since either YPT1 or SLY1-20 can suppress both mutations in, and a deletion of, SEC34, yet overexpression of SEC34 cannot suppress mutations in either YPT1 or SLY1, we hypothesize that Ypt1p and Sly1p function downstream of Sec34p.
Weaker suppression of the sec34-2
mutation was observed upon overexpression of the tethering factor Uso1p, or the v-SNAREs Sec22p, Bet1p, or Ykt6p. The suppression of sec34-2
by the v-SNAREs may be through mass action, in which vesicles containing supernumerary v-SNAREs are able to compensate for a deficiency in tethering, albeit with a very low efficiency. This phenomena has been observed previously for mutations in the tethering factors Uso1p ( Sapperstein et al. 1996
) and Sec35p ( VanRheenen et al. 1998
), as well as components of the putative tethering complex TRAPP ( Jiang et al. 1998
). Interestingly, no suppression of the sec34-2
mutation was observed upon overexpression of either the v-SNARE Bos1p or the cis-Golgi complex t-SNARE Sed5p. Overexpression of Bos1p was also unable to suppress a temperature-sensitive growth defect of the sec35-1
strain ( VanRheenen et al. 1998
) or the inviability of the uso1
Δ strain ( Sapperstein et al. 1996
). This lack of suppression could result from inefficient expression of the gene or may indicate a functional difference between Bos1p and the other v-SNAREs that mediate the ER to Golgi complex transport step. The lack of suppression of the sec34-2
mutant strain by high-copy expression of Sed5p was not unexpected, because it has been demonstrated that overexpression of this t-SNARE is toxic to cells ( Hardwick and Pelham 1992
Biochemical analysis of the Sec34 protein reveals that it is a peripheral membrane protein. Although a small amount of the protein is soluble, the remainder partitions between the P10 and P175 fractions, similar to the Golgi protein Sed5p; it is possible, therefore, that Sec34p is associated with the Golgi complex. Due to the association of Sec34p with membranes, we used semi-intact cells made from the sec34-2 strain to test the requirement for Sec34p in tethering through an assay that reconstitutes ER to Golgi complex transport. These semi-intact cells were demonstrated to be able to bud vesicles from the ER, but these vesicles failed to efficiently tether to the Golgi complex at the restrictive temperature, indicating that Sec34p is required for the tethering of ER-derived vesicles to the cis-Golgi complex. Since cytosolic proteins are removed from the sec34-2 semi-intact cells, the membrane-associated pool of Sec34-2p is most likely the source of the tethering defect. In addition, since the membranes involved in tethering are restricted to those of the vesicle and the cis-Golgi complex, Sec34p is most likely associating with one, or both, of these membranes.
was found to display two interesting genetic interactions with the tethering factor gene SEC35
. First, multicopy SEC34
weakly suppresses a temperature-sensitive allele of SEC35
. Since overexpression of SEC34
cannot suppress the cold-sensitive lethality of the sec35
Δ strain, Sec34p is able to assist a handicapped allele of SEC35
, but cannot replace its function. Second, the sec34-2
alleles display a synthetic lethal interaction. Although strains bearing either allele alone are permissive for growth at 23 and 30°C, a haploid strain containing both the sec34-2
alleles is inviable at either temperature. This synthetic phenotype is more severe than the conditional synthetic lethality of the sec35-1
allele in combination with a mutant allele of either YPT1
, in which the double mutants are viable at 23°C, but not at 30°C ( VanRheenen et al. 1998
). This finding suggests a close functional interaction of the Sec34 and Sec35 proteins.
Based on these results, we investigated whether Sec34p and Sec35p could physically interact through the two-hybrid assay. Indeed, Sec34p and Sec35p were found to interact. The interaction between the two proteins may explain the ability of multicopy SEC34 to suppress the sec35-1 allele, but not the sec35Δ allele: increased levels of Sec34p could stabilize a defective form of Sec35p but would be ineffectual in the absence of Sec35p, especially if the interaction of the two proteins is essential to their function in tethering. To further explore the interaction of Sec34p and Sec35p we examined the behavior of the soluble pool of these proteins through several chromatographic steps. The proteins cofractionated through ammonium sulfate precipitation and anion exchange, cation exchange, ceramic hydroxyapatite, and size exclusion chromatographic steps, providing strong evidence that the two proteins are in a complex with one another. Intriguingly, the Sec34p/Sec35p complex appears quite large, with an estimated molecular weight (if globular) of ~750 kD. This size, which is larger than the combined molecular weights of the two proteins (124 kD), suggests several possibilities for the structure of the complex. First, the complex could be homodimeric, containing one molecule of each protein, but highly elongated such that it migrates rapidly through a size exclusion column. We consider this unlikely because the sequences of Sec34p and Sec35p lack motifs (such as coiled-coil domains) that would be indicative of an elongated structure. Second, the complex could contain two or more molecules of at least one protein, resulting in a more massive structure. Finally, the complex could be multimeric, containing heretofore unidentified component(s) in addition to Sec34p and Sec35p. We are currently purifying the Sec34p/Sec35p complex to address this issue and identify any additional components. It appears, however, that Uso1p is unlikely to be a component of the Sec34p/Sec35p complex since immunoblotting fractions from the purification with an antibody against this protein revealed that Uso1p did not comigrate with Sec34p and Sec35p (data not shown).
The 750-kD complex containing Sec34p and Sec35p is reminiscent of the TRAPP complex, which migrates at ~800 kD by size exclusion chromatography ( Sacher et al. 1998
). However, two pieces of data indicate that the TRAPP complex is distinct from the Sec34p/Sec35p complex. First, the identities of the low molecular weight members of the TRAPP complex have been elucidated, and none corresponds to Sec35p, whose mobility on SDS-PAGE was within the range of the proteins that have been sequenced ( Sacher et al. 1998
). In addition, the known members of the TRAPP complex display genetic interactions with one another ( Jiang et al. 1998
; Sacher et al. 1998
), yet no interaction was discerned between the gene encoding the TRAPP component Bet3p and either SEC34
(this work and VanRheenen et al. 1998
Since many secretory factors are evolutionarily conserved, we explored whether the components of the Sec34p/Sec35p complex were conserved in higher eukaryotes. The genome of the nematode C. elegans was discovered to contain a protein designated Y71F9A 290.A that is very similar to Sec34p. However, the C. elegans protein is ~50% the size of Sec34p and therefore may not be a true ortholog. We also discovered a C. elegans protein with moderate homology to Sec35p (22% identical and 33% similar), designated C35A5.6. While the similarity is not high, the proteins are similar in size (C35A5.6 is comprised of 273 amino acid residues, whereas Sec35p is comprised of 275 amino acid residues), and thus, this C. elegans protein is a putative ortholog of Sec35p. Searches of GenBank for additional homologs of these proteins did not reveal additional Sec35p homologs, but several human ESTs were discovered with a high degree of similarity to Sec34p. Interestingly, the sequences contained on these ESTs were homologous to Sec34p over only a portion of the analyzed region of the putative human protein, and thus the protein may contain a Sec34p-like domain and may not be a true Sec34p ortholog. These data indicate that there may be orthologs of the Sec34p/Sec35p complex in higher organisms, but functional experiments will be required to unambiguously address this point. Finally, a putative ortholog of Sec34p was discovered in the genome of S. pombe. No paralogs of either Sec34p or Sec35p exist in S. cerevisiae, and thus these proteins do not define a family of related proteins.
Finally, we describe the identification and characterization of a gene designated RUD3
that displays a genetic interaction with SEC34
, which encodes a novel nonessential protein with a predicted molecular weight of 56 kD, was originally identified in a screen for multicopy suppressors of a temperature-sensitive allele of the tethering factor, USO1
( Sapperstein et al. 1996
; Sapperstein 1997
), and is also able to suppress the temperature-sensitive growth defect of the sec34-2
strain. Interestingly, RUD3
is unable to suppress mutations in other ER to Golgi complex docking factors such as Sec35p, Ypt1p, Sec22p, Bet1p, or Bos1p, and thus the suppression is specific to mutant alleles of SEC34
. Overexpression of RUD3
can weakly suppress the inviability of the uso1
Δ strain (data not shown). Taken together, these data suggest that Rud3p either acts at, or downstream of, the tethering stage of ER to Golgi complex transport. Rud3p does not appear to be a component of the Sec34p/Sec35p complex since the majority of the protein fractionates away from the complex during its purification (data not shown).
In summary, we describe the characterization of a novel secretory factor, Sec34p, and its role in tethering of ER-derived vesicles to the cis-Golgi complex. Unlike the SNAREs and rabs, the tethering factors described thus far at different intracellular transport steps are not members of a protein family. Nevertheless, they do share structural similarity, since they are either elongated or present in a large multimeric complex ( Pfeffer 1999
; Waters and Pfeffer 1999
). The large size may be related to the requirement for the tethering factors to span the distance between the vesicle and the target compartment, before trans-SNARE complex formation. Interestingly, three factors meet this criteria in the yeast ER to Golgi complex transport step: the extended homodimer Uso1p, the TRAPP complex, and the Sec34p/Sec35p complex. It will be very exciting to discover in the future how these large protein complexes function to secure a vesicle to its target membrane, and whether their function is more complex than simply connecting vesicle and target membranes.