We have identified a novel class of Sec6p mutants that disrupt the polarized localization of the exocyst complex at nonpermissive conditions, yet do not affect complex assembly. We show that mutation of conserved surface residues on the C-terminal helical bundle domain of Sec6p do not destabilize full-length Sec6p (), yet result in severe temperature- and media-sensitive growth defects (). At the nonpermissive conditions, vesicle trafficking to sites of secretion appears unimpaired, whereas the Sec6p patch mutants are completely mislocalized (). Consequently, all of the other exocyst subunits are also mislocalized (). Previous studies of exocyst temperature sensitive mutants showed that this mislocalization is caused by destabilization and disruption of the exocyst complex. However, we show that exocyst complexes remain intact in the sec6 patch mutant strains, even at the nonpermissive conditions ().
Unexpectedly, the extensive exocyst mislocalization observed in our sec6-49
strains at the nonpermissive conditions included Sec3p, which was proposed to be the spatial landmark for exocyst localization (Finger et al., 1998
). Sec3p-GFP appeared polarized in yeast cells in the absence of ongoing exocytosis and in other exocyst ts mutants (Finger et al., 1998
; Zhang et al., 2005b
). However, localization of the endogeneous Sec3p by immunofluorescence indicated that the GFP tag may artificially stabilize Sec3p-GFP at sites of secretion (Roumanie et al., 2005
), although this was recently disputed (Zhang et al., 2008
). Localization of the endogenous Sec3p in sec6-4
cells (Roumanie et al., 2005
) was similar to our results with Sec3-GFP in the sec6-4
strain. Several variations between our studies and previous results could account for these discrepancies: the use of plasmid-borne, instead of genomic GFP-tagged Sec3p; the testing of Sec3-GFP in the presence of the endogenous Sec3p; differences in strain backgrounds; and the fact that our construct contains a linker sequence (9 a.a.) between the C-terminus of Sec3p and the GFP (Huh et al., 2003
). Our data, combined with the observations that Sec3p is not an essential gene and that the other exocyst subunits are polarized in sec3
Δ cells at 25°C (Wiederkehr et al., 2003
; Zhang et al., 2005b
), suggest that Sec3p is not the spatial landmark for the exocyst complex.
Other candidates for targeting the exocyst to sites of secretion are Exo70p and Exo84p (Boyd et al., 2004
; He et al., 2007
). However, we found that Exo70p-GFP was mislocalized in the sec6-49
strains at the nonpermissive conditions (). Similarly, although the localization of Exo84-GFP was proposed to be independent of the exocyst subunits (e.g., not mislocalized in previous ts strains; Zhang et al., 2005b
), Exo84-GFP was also mislocalized in the sec6-49
strains (). Therefore, we conclude that the conserved surface patches on Sec6p are required for localization and/or stability of the exocyst complex at sites of secretion and that Sec3p, Exo70p, and Exo84p are not sufficient to localize the complex in these sec6
mutant strains. Nevertheless, the presence of a landmark is not strictly necessary, because actin cables are polarized independently of the exocyst, bringing vesicles to the correct sites of exocytosis (Walch-Solimena et al., 1997
). Therefore, we propose that exocyst subunits travel to sites of secretion where they assemble together, and that Sec6p is required to anchor the assembled exocyst complexes at these sites. Perhaps Sec6p acts in cooperation with Sec3p, Exo70p, and/or Exo84p for stabilization of the exocyst complexes on the plasma membrane or perhaps their interactions (e.g., with Rho proteins and phosphoinositides) are more regulatory in nature (Roumanie et al., 2005
; Wu et al., 2008
). It may be that either Exo70p or Sec3p are sufficient to localize the exocyst, when wild-type Sec6p is present to stabilize the complex at sites of secretion (Hutagalung et al., 2008
The spatial and temporal regulation of exocyst assembly and disassembly is currently unclear. Most of the exocyst subunits appear to traffic to the plasma membrane with secretory vesicles (Boyd et al., 2004
), although it is unknown whether they traffic as preassembled complexes or they assemble upon vesicle arrival. Assembly upon arrival at sites of secretion, which was suggested by FRAP data (Boyd et al., 2004
), is compelling, because the interactions between subunits on the vesicle with those on the plasma membrane could tether the membranes together. If so, then the patch residues of wild-type Sec6p would be necessary to anchor the complex at these sites after vesicle arrival. In the case of the patch mutants, Sec4p would need to hydrolyze the bound GTP, perhaps upon exocyst assembly, to release Sec15p and therefore the rest of the exocyst complex. On the other hand, it is possible that the exocyst assembles at sites of vesicle formation and budding; the patch residues of Sec6p would be required for proper trafficking of the complex to sites of secretion. This could only be the case if vesicles are capable of transport in the absence of assembled exocyst complexes, because trafficking of vesicles containing Sec4p is unimpaired in the patch mutant strains (as well as in the sec6-4
and other exocytic sec
mutants). This situation would require that Sec4p on the budding vesicle hydrolyze the bound GTP to release the complex. Further mutational and GFP localization studies will be necessary to distinguish between these scenarios.
Regardless of where assembly takes place, the finding that the complex remains assembled despite a block in exocytosis and in the absence of polarized accumulation suggests two possibilities. First, the complex cannot disassemble, because disassembly is triggered by a factor(s) at sites of secretion or by membrane fusion. If the complex is locked into an assembled conformation in the sec6-49 and -54 mutants, then it may be unable to be recycled for subsequent rounds of vesicle tethering and fusion, thus contributing to the ts phenotype. Alternatively, the complex is assembled in these mutants because it normally does not disassemble unless destabilized. In this case, mislocalization of the assembled exocyst complexes at nonpermissive conditions would lower the concentration of active exocyst complexes that are available to function at sites of secretion. This would ultimately result in decreased SNARE complex assembly and membrane fusion.
Conservation of these Sec6p patch residues suggests an important anchoring function for Sec6p in all eukaryotes. Our analyses indicate that the binding partner for the patches on Sec6p is not a subunit of the exocyst complex, nor is it the t-SNARE Sec9p (). Therefore, further genetic and biochemical experiments will be necessary to identify the anchoring factor and analyze its role in polarizing Sec6p and the exocyst complex at sites of secretion.