In a prior study, we had found that the localization of Sec3p to bud tips and necks is maintained even when vesicular transport is blocked or when the actin cytoskeleton is depolymerized (Finger et al., 1998
). Sec3p was therefore proposed to act as a spatial landmark for polarized secretion. The findings presented here strongly support a model in which Sec3p marks subdomains of the plasma membrane for efficient targeting and tethering of secretory vesicles. Furthermore, we now extend that proposal to include a role for Sec3p, and perhaps other components of the exocyst, as an anchor at the bud tip for ER tubules segregated into daughter cells.
Here, we show that, unlike all other subunits of the exocyst, Sec3p is not essential for growth or for export of secretory cargo. Furthermore, the pattern of actin assembly as well as the actin-dependent polarized transport of secretory vesicles and other organelles looks normal in sec3Δ cells. Indeed, the vesicle marker Sec4p and the GFP-tagged exocyst subunit Sec8p are still found concentrated in the bud or close to the mother bud neck under permissive growth conditions. However, in the absence of Sec3p, the vesicles do not seem to find appropriate exocytic sites within the bud, and this results in the fusion of vesicles along the entire surface of the bud. This hypothesis is supported by the broader distribution of Sec4p and the more diffuse localization of Sec8-GFP within the buds of sec3Δ cells relative to wild-type buds (Figures and ). Such delocalized fusion within the bud would lead to isotropic growth and hence explain the observed round morphology of sec3Δ cells. A similar situation occurs during the formation of mating projections in response to mating factor. Vesicles marked by Sec4p are still delivered in a polarized manner in sec3Δ cells, but rather than fusing within a well focused spot, they fuse along a broad front and thereby give rise to a bulbous mating projection. Inefficient targeting may also lead to the failure of a fraction of the vesicles to fuse, as seen in sec3Δ cells under permissive conditions. At 37°C, sec3Δ cells, similar to conditional mutants defective in other exocyst components, are tightly blocked for growth, fail to export a large fraction of their secretory cargo and accumulate secretory vesicles in a polarized manner. In addition, the polarized localization of Sec8-GFP is lost after a shift of sec3Δ cells to 37°C (). Sec3p may be critical for the stability of the exocyst at 37°C, whereas at 25°C the complex may remain sufficiently assembled or active to mediate its essential functions in exocytosis without Sec3p. Thus, a critical function of Sec3p may be to bind and stabilize the exocyst complex at exocytic sites.
We also report the unexpected finding that Sec3p plays a key role in the inheritance of cortical ER. In S. cerevisiae
, this seems to be a multistage process involving transport of cytoplasmic ER tubules along the mother-bud axis, followed by their association with the bud tip and then their propagation along the bud cortex (Du et al., 2001
) (Fehrenbacher et al., 2002
; our unpublished data). The daughter cell obtains ER tubules at the cortical region early in S phase, shortly after the initiation of bud growth (Preuss et al., 1991
; Du et al., 2001
). In contrast, more than half of sec3
Δ mutant buds fail to acquire ER tubules at their cortex even at late S to G2 phases (). Moreover, a significant fraction of sec3
Δ cells contains tubules across the bud as the only inherited ER elements. Thus, we speculate that Sec3p marks the bud tip destination for ER tubules, similar to its role as a landmark for secretory vesicles. Electron microscopy revealed that many of the sec3
Δ cells had an unusually large amount of cytoplasmic ER in the mother cell, in most cases close to the nucleus (), and a similar phenotype had been noted in a study of sec3-4
mutant cells (Finger and Novick, 1997
). Because ER to Golgi transport is unaffected in these cells, it is possible that the structures represent ER tubules that have been retracted back into the mother cell after an unsuccessful attempt to associate with the cortex of the bud. Time-lapse microscopy shows that ER tubules are highly dynamic in sec3
Δ cells. Tubules move across the neck, but fail to be stably retained in the bud. Neither mitochondria nor late Golgi, two other organelles that are transported to the bud tip during the cell cycle (Simon et al., 1997
; Rossanese et al., 2001
), require the function of Sec3p for proper inheritance (). It therefore seems that the role of Sec3p is limited to the localization of secretory vesicles and ER tubules.
Rho1p acts through at least two pathways to control the localization of the exocyst. The amino terminus of Sec3p has been shown to bind to Rho1p (Guo et al., 2001
) and an allele of Sec3p deleted for the Rho1p-binding domain (Sec3ΔN-GFP) is mislocalized, but only when expressed in a wild-type SEC3
background. When expressed as the sole copy, partial localization of Sec3ΔN-GFP is seen and the other subunits of the exocyst still localize quite well (Guo et al., 2001
) implying a Sec3-independent pathway for exocyst localization. Apparently, the truncated allele of Sec3p can localize to some extent by assembling with the rest of the exocyst. We show here that expression of Sec3ΔN is able to complement the ER inheritance defect of a sec3
Δ strain. A temperature-sensitive rho1
mutant does show a partial defect in ER inheritance after a shift to the restrictive temperature (our unpublished observations). A more complete defect is not seen because this allele may be only partially defective for this function, because other Rho family members are partially redundant or because some of the buds contain ER that had been segregated before the temperature shift. In general, it is difficult to assay organelle inheritance in mutants such as rho1
that are blocked in bud growth.
An important clue to the role of Sec3p in ER inheritance has come from our observation of the effects of Sec3p overexpression. The formation of a prominent patch of Sec61-GFP at the bud tip in response to Sec3p overexpression, without a parallel change in Hmg1-GFP localization or an accumulation of ER membrane suggests that the patch may represent a selective enrichment of Sec61-GFP within the plane of the ER membrane at the bud tip, rather than an actual increase in the amount of ER membrane at this site. Such a selective enrichment could reflect a physical linkage, either direct or through intermediaries, between Sec61-GFP and the exocyst. Both genetic as well as physical interactions have been observed between the exocyst and components of the translocon. SEB1
, encoding the β subunit of the Sec61 translocon complex, was identified as a high copy number suppressor of sec15-1
(Toikkanen et al., 1996
) and was recently shown to suppress defects in the other subunits of the exocyst as well. Overexpression of the other two subunits of the translocon suppresses several exocyst mutants. Coprecipitation experiments indicate a physical linkage between the exocyst and the translocon in both yeast (Toikkanen et al., 2003
) and mammalian cells (Lipschutz et al., 2003
). In total, these studies support the proposed physical connection between the exocyst at the apical tip of the plasma membrane and the Sec61p-containing translocon complex on the ER membrane. Although the efficiency of coprecipitation reported was not high, it is consistent with the observation that only a small portion of the ER is actually anchored at the bud tip and that both the translocon and exocyst complexes have other essential functions. Establishing the details of this interaction between two large, multisubunit complexes will require further studies.
Together, our results show that Sec3p is not essential for exocytosis in yeast but is required for tethering secretory vesicles to specialized subdomains of the plasma membrane. In a similar manner, Sec3p is also required to stabilize the cortical association of ER tubules that have been extended into the yeast bud. In this way, Sec3p may initiate ER retention and promote distribution along the cortex of the newly forming bud. The Sec3p mediated connection between cortical ER and exocytosis at the bud tip together with the actin-mediated inheritance of Golgi structures may establish an autonomous secretory apparatus in the daughter cells soon after bud initiation and thereby facilitate polarized growth.