Reprogramming nucleolar size by genetic perturbation of the extranuclear Rab GTPases Ypt6 and Ypt32.

Reprogramming organelle size has been proposed as a potential therapeutic approach. However, there have been few reports of nucleolar size reprogramming. We addressed this question in Saccharomyces cerevisiae by studying mutants having opposite effects on the nucleolar size. Mutations in genes involved in nuclear functions (KAR3, CIN8, and PRP45) led to enlarged nuclei/nucleoli, whereas mutations in secretory pathway family genes, namely the Rab-GTPases YPT6 and YPT32, reduced nucleolar size. When combined with mutations leading to enlarged nuclei/nucleoli, the YPT6 or YPT32 mutants can effectively reprogram the nuclear/nucleolar size almost back to normal. Our results further indicate that null mutation of YPT6 causes secretory stress that indirectly influences nuclear localization of Maf1, the negative regulator of RNA Polymerase III, which might reduce the nucleolar size by inhibiting nucleolar transcript enrichment.

Yeast dynamin and Ypt6 function in parallel for the endosome-to-Golgi retrieval of Snc1.

Protein recycling is an important cellular process required for cell homeostasis. Results from prior studies have shown that vacuolar sorting protein-1 (Vps1), a dynamin homolog in yeast, is implicated in protein recycling from the endosome to the trans-Golgi Network (TGN). However, the function of Vps1 in relation to Ypt6, a master GTPase in the recycling pathway, remains unknown. The present study reveals that Vps1 physically interacts with Ypt6 if at least one of them is full-length. We found that overexpression of full-length Vps1, but not GTP hydrolysis-defective Vps1 mutants, is sufficient to rescue abnormal phenotypes of Snc1 distribution provoked by the loss of Ypt6, and vice versa. This suggests that Vps1 and Ypt6 function in parallel pathways instead of in a sequential pathway and that GTP binding/hydrolysis of Vps1 is required for proper traffic of Snc1 toward the TGN. Additionally, we identified two novel Vps1-binding partners, Vti1 and Snc2, which function for the endosome-derived vesicle fusion at the TGN. Taken together, the present study demonstrates that Vps1 plays a role in later stages of the endosome-to-TGN traffic.

Small GTPase proteins in macroautophagy.

Macroautophagy, a highly conserved process in eukaryotic cells, is initiated in response to stress, especially nutrient starvation. Macroautophagy helps cells survive by engulfing proteins and organelles into an unusual double-membraned structure called the autophagosome, which then fuses with the lysosome. Upon degradation of the engulfed contents, the building blocks are recycled for synthesis of new macromolecules. Recent work has demonstrated that construction of the autophagosome requires a variety of small GTPases in variations of their normal roles in membrane traffic. In this Commentary, we review our own recent findings with respect to 2 different GTPases, Arl1, a member of the Arf/Arl/Sar family, and Ypt6, a member of the Rab family, in the yeast S. cerevisiae in light of other information from the literature and discuss future directions for further discerning the roles of small GTPases in autophagy.

A High Copy Suppressor Screen for Autophagy Defects in Saccharomyces arl1Δ and ypt6Δ Strains.

In Saccharomyces cerevisiae, Arl1 and Ypt6, two small GTP-binding proteins that regulate membrane traffic in the secretory and endocytic pathways, are also necessary for autophagy. To gain information about potential partners of Arl1 and Ypt6 specifically in autophagy, we carried out a high copy number suppressor screen to identify genes that when overexpressed suppress the rapamycin sensitivity phenotype of arl1Δ and ypt6Δ strains at 37°. From the screen results, we selected COG4, SNX4, TAX4, IVY1, PEP3, SLT2, and ATG5, either membrane traffic or autophagy regulators, to further test whether they can suppress the specific autophagy defects of arl1Δ and ypt6Δ strains. As a result, we identified COG4, SNX4, and TAX4 to be specific suppressors for the arl1Δ strain, and IVY1 and ATG5 for the ypt6Δ strain. Through this screen, we were able to confirm several membrane traffic and autophagy regulators that have novel relationships with Arl1 and Ypt6 during autophagy.

Autophagy in Saccharomyces cerevisiae requires the monomeric GTP-binding proteins, Arl1 and Ypt6.

Macroautophagy/autophagy is a cellular degradation process that sequesters organelles or proteins into a double-membrane structure called the phagophore; this transient compartment matures into an autophagosome, which then fuses with the lysosome or vacuole to allow hydrolysis of the cargo. Factors that control membrane traffic are also essential for each step of autophagy. Here we demonstrate that 2 monomeric GTP-binding proteins in Saccharomyces cerevisiae, Arl1 and Ypt6, which belong to the Arf/Arl/Sar protein family and the Rab family, respectively, and control endosome-trans-Golgi traffic, are also necessary for starvation-induced autophagy under high temperature stress. Using established autophagy-specific assays we found that cells lacking either ARL1 or YPT6, which exhibit synthetic lethality with one another, were unable to undergo autophagy at an elevated temperature, although autophagy proceeds normally at normal growth temperature; specifically, strains lacking one or the other of these genes are unable to construct the autophagosome because these 2 proteins are required for proper traffic of Atg9 to the phagophore assembly site (PAS) at the restrictive temperature. Using degron technology to construct an inducible arl1Δ ypt6Δ double mutant, we demonstrated that cells lacking both genes show defects in starvation-inducted autophagy at the permissive temperature. We also found Arl1 and Ypt6 participate in autophagy by targeting the Golgi-associated retrograde protein (GARP) complex to the PAS to regulate the anterograde trafficking of Atg9. Our data show that these 2 membrane traffic regulators have novel roles in autophagy.

The TRAPP Subunit Trs130p Interacts with the GAP Gyp6p to Mediate Ypt6p Dynamics at the Late Golgi.

Small GTPases of the Rab superfamily participate in virtually all vesicle-mediated trafficking events. Cycling between an active GTP-bound form and an inactive GDP-bound form is accomplished in conjunction with guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively. Rab cascades have been described in which an effector of an activated Rab is a GEF for a downstream Rab, thus ensuring activation of a pathway in an ordered fashion. Much less is known concerning crosstalk between GEFs and GAPs although regulation between these factors could also contribute to the overall physiology of a cell. Here we demonstrate that a subunit of the TRAPP II multisubunit tethering factor, a Rab GEF, participates in the recruitment of Gyp6p, a GAP for the GTPase Ypt6p, to Golgi membranes. The extreme carboxy-terminal portion of the TRAPP II subunit Trs130p is required for the interaction between TRAPP II and Gyp6p. We further demonstrate that TRAPP II mutants, but not a TRAPP III mutant, display a defect in Gyp6p interaction. A consequence of this defective interaction is the enhanced localization of Ypt6p at late Golgi membranes. Although a ypt31/32 mutant also resulted in an enhanced localization of Gyp6p at the late Golgi, the effect was not as dramatic as that seen for TRAPP II mutants, nor was Ypt31/32 detected in the same TRAPP II purification that detected Gyp6p. We propose that the interaction between TRAPP II and Gyp6p represents a parallel mechanism in addition to that mediated by Ypt31/32 for the recruitment of a GAP to the appropriate membrane, and is a novel example of crosstalk between a Rab GAP and GEF.

Analysis of subcellular localization and function of the yeast Rab6 homologue, Ypt6p, using a novel amino-terminal tagging strategy.

Ypt6p, the yeast homologue of mammalian Rab6, is involved in the multiple processes regulated by membrane trafficking such as vacuole maturation and membrane protein recycling. Although several lines of evidence suggest that Ypt6p is possibly localized to multiple membrane compartments, the precise localization of endogenous Ypt6p remains to be elucidated. In this study, we developed a novel method for N-terminal tagging of endogenous protein based on homologous recombination and investigated the subcellular localization and function of Ypt6p. Ypt6p and its GTP-bound form were predominantly localized to the cis- to medial-Golgi compartments whereas the GDP-bound form of Ypt6p was localized to the cytosol. Ric1p, a component of the specific GEF complex for Ypt6p, largely colocalized with Ypt6p in the early Golgi, and localization of Ypt6p changed to the cytosol in ric1Δ cells. On the other hand, Gyp6p, a putative GAP for Ypt6p, was localized to the trans-Golgi compartment and deletion of GYP6 increased the localization of Ypt6p at the trans-Golgi, suggesting that Gyp6p promotes the dissociation of Ypt6p from the Golgi when arriving at the trans-Golgi compartment. Additionally, we demonstrated that overexpression of the GDP-bound form of Ypt6p caused defective vacuole formation and recycling of Snc1p to the plasma membrane. These results suggest that the GTP-binding activity of Ypt6p is necessary for intra-Golgi trafficking and protein recycling in the early Golgi compartment.