Sec4p, the Rab protein required for post-Golgi transport in budding yeast, is associated with secretory vesicles (Goud et al., 1988). Sec2p is the essential nucleotide exchange factor for Sec4p and is required for the polarized delivery of Sec4p-vesicles to exocytic sites. Sec2p activates Sec4p by increasing both the GDP off-rate and the binding of GTP, thus promoting the interaction of GTP-Sec4p with effectors required for vesicular transport (Walch-Solimena). In this study, we establish a role for the COOH terminus of Sec2p in the efficient polarized delivery of Sec4p vesicles.
Previous work has shown that COOH-terminal truncation of Sec2p produces a defective protein causing a temperature-sensitive growth and secretion phenotype (Nair et al. 1990
). However, the results presented here show that loss of the COOH terminus of Sec2p does not reduce its nucleotide exchange activity (GTP on-rate) to the extent that would cause temperature sensitivity. In support of this, we expressed levels of full-length Sec2p that were undetectable by Western blot, with no resulting growth defect. These cells would have a much greater decrease in Sec2p function than the ~20% decrease in exchange activity observed for the truncated protein Sec2-59(1-374)-GFP. Thus, the COOH terminus must have another function.
Full-length Sec2-GFP localizes to sites of exocytosis, including the presumptive bud site, small buds and the mother/daughter neck (). A 58–amino acid domain, present in the COOH terminus, was shown to be necessary but not sufficient for this localization. Sec2 proteins, either lacking the 58–amino acid domain, or possessing a point mutation within this domain (sec2-78) are not concentrated at sites of exocytosis. In addition, this domain is required for the tight membrane association displayed by full-length and not truncated Sec2 proteins. The localization of Sec2p to sites of exocytosis correlates with its membrane association.
The localization of Sec2p to exocytic sites is reminiscent of Sec4p localization (Novick and Brennwald 1993
; Walch-Solimena et al. 1997
), and is similar to that observed for other late-acting secretory proteins functioning at the sites of polarized growth; namely, Sec5p (Mondesert et al. 1997
), Sec3p (Finger et al. 1998
), Sec8 (TerBush and Novick 1995
), Sec1p (Carr et al. 1999
), and Exo84p (Guo et al. 1999a
). As in the case of Sec4p, the localization of Sec2-GFP to these sites requires the ongoing production of secretory vesicles as well as the function of the actin-based cytoskeleton and the Myo2p motor. We interpret this localization to reflect the association of Sec2-GFP with secretory vesicles that are concentrated at sites of exocytosis in an actin-dependent fashion. There are several situations in which the localization of Sec2-GFP differs from that of Sec4p. Sec4p localization is independent of all of the late acting Sec proteins except Sec2p, while Sec2-GFP requires, in addition to Sec4p function, the function of Sec1p, Sec6p, and Sec9p as well. We postulate that Sec2p normally recycles after activating Sec4p. Mutants defective in Sec1p, Sec6p, and Sec9p function are blocked after Sec2p has recycled, leading to an accumulation of vesicles carrying Sec4p but not Sec2-GFP. Other mutants are blocked before this event, leading to the accumulation of vesicles carrying both Sec4p and Sec2-GFP. In support of this hypothesis we have shown that membrane association of full-length Sec2-GFP is perturbed in both sec6-4
mutant cells at the restrictive temperature, but not in sec3-2
We propose that Sec2p associates with secretory vesicles and this allows its nucleotide exchange activity greater accessibility to Sec4p, whose activation is required for vesicle transport. When Sec2p membrane attachment is compromised and the activation of Sec4p occurs less efficiently (as in a sec2(1-450), sec2-59(1-374),
mutant cells), vesicles accumulate in a random fashion. Yeast overexpressing truncated sec2
alleles bypass the need for Sec2p membrane attachment and thereby overcome this growth defect. In our study this increase in expression occurs when sec2(1-450)
, or sec2-78
are fused to GFP
and integrated at the LEU2
locus. Sec2 proteins defective for membrane attachment may still interact with Sec4p during transport, however, we propose that this interaction is partially compromised. This explains the detection of both Sec2-59(1-374)p and Sec2-78p in association with the vesicular pool in previous work from our lab (Walch-Solimena et al. 1997
). It is also possible that the interaction of truncated Sec2 proteins with Sec4p is able to withstand sucrose, as was used in the earlier study (Walch-Solimena et al. 1997
), but not iodixanol gradient centrifugation, as used here.
If Sec2p function is required for efficient vesicle targeting, Sec4p activation may be required for the transport event and/or upon arrival at sites of exocytosis. Analogous to Rab 5 function during endosome fusion (Rybin et al. 1996
), Sec4p may also undergo continuous cycles of exchange during transport in order to ensure that it is in its activated GTP-bound form, ready to interact with its docking effector, Sec15p (Guo et al. 1999b
), upon reaching the destination. To date, Sec15p is the only effector known for Sec4p and sec15-1
is not defective for the polarized delivery of vesicles to exocytic sites. We therefore anticipate the existence of an additional Sec4p effector that would serve to couple Sec4 activation to the vesicle transport machinery. This effector would be analogous to the Golgi-associated kinesin-like effector for Rab6 which plays a role in Golgi dynamics in mammalian cells (Echard et al. 1998
Vectorial transport of post-Golgi vesicles in yeast is mediated by the actin cytoskeleton. Post-Golgi vesicles accumulate randomly in LAT-A–treated cells (Karpova et al. 2000
), reminiscent of the accumulation of vesicles in an act1
mutant background (Novick and Botstein 1985
; Walch-Solimena et al. 1997
). Indirect evidence has implicated the unconventional type V myosin, Myo2p, as the motor involved in the transport process in yeast since mutations in MYO2
, or overexpression of the Myo2p-tail domain, lead to a random accumulation of vesicles (Govindan et al. 1995
; Karpova et al. 2000
; Reck-Peterson et al. 1999
; Schott et al. 1999
). Accordingly, Sec4p (Walch-Solimena et al. 1997
) and Sec2p ( and text) are mislocalized in myo2-66
. Sec2-GFP is partially mislocalized in myo2-2
. This allele specifically affects vacuolar inheritance and has no growth phenotype and no defect in Sec4p localization. The myo2-2
mutation is in the tail domain and Myo2-2p is severely impaired for vacuole binding. This mutation might also affect the interaction between Myo2-2p and vesicles to some extent as well. In this case, vesicle transport may be mildly affected to the point where Sec2p is partially mislocalized but with no effect on growth rate.
In mammalian cells, filamentous actin, organized at the cell periphery, may play a role in short-range movement or capture of vesicles transported there via a long-range microtubule-based mechanism (Wu et al. 1998
). In yeast microtubules are not required for post-Golgi vectorial transport. In dilute
mice, a defective myosin Va leads to a perinuclear distribution of melanocytes causing a lighter colored coat. Normal melanocytes possess randomly distributed melanosomes which cause the normal, darker, coat color (Provance et al. 1996
; Wei et al. 1997
). These results and the visualization of melanosome dynamics in dilute
melanocytes (Wu et al. 1998
) imply that myosin Va may act to tether these vesicles to the cell periphery. In support of this, disruption of actin in melanoma cells leads to a phenotype similar to that observed in dilute
melanocytes (Koyama and Takeuchi 1980
). In humans, myosin Va may be the gene involved in Griscelli's syndrome, where patients have a diluted pigment as well as immunodeficiency and/or neurological phenotypes (Griscelli et al. 1978
; Haraldsson et al. 1991
; Hurvitz et al. 1993
; Klein et al. 1994
; Pastural et al. 1997
). Studying the actin-mediated transport of vesicles in yeast may shed light on the interactions between Rab proteins and unconventional myosins in transport.
In conclusion, we have characterized a 58–amino acid domain in the COOH terminus of Sec2p that regulates the association of Sec2p with membranes. Sec2p membrane association is correlated with a polarized localization, reminiscent of Sec4p localization. The data presented here are consistent with a model where Sec2p is carried on post-Golgi vesicles, activating Sec4p either before or during transport. This implies the existence of an unidentified Sec4p transport effector. This effector may be part of a transport complex that links post-Golgi vesicles to the actin cytoskeleton via the associated motor Myo2p. Elucidating the molecular machinery linking vesicles to cytoskeletal systems will shed light on the mechanisms responsible for movement and localization of post-Golgi vesicles.