Irs4p and Tax4p: two redundant EH domain proteins involved in autophagy.

Proteins carrying EPS15 homology (EH) domains are present from yeast to mammals. The characterized members of this protein family are all involved in intracellular trafficking, typically endocytosis and endocytic recycling. We focused on two members of this family in Saccharomyces cerevisiae Irs4p and Tax4p, whose functions are less well characterized. We show that the deletion of IRS4 altered the function of a neighboring gene, VPS51, involved in endocytic recycling. The irs4Deltatax4Delta cells complemented for the loss of Vps51p (irs4Deltatax4Delta*) display no defects in endocytosis and endosomal recycling, clearly differentiating these two EH proteins from the other protein family members. Because Irs4p is phosphorylated when autophagy is induced, we studied the potential role of these two proteins in this latter process. We observed a loss of viability upon starvation in irs4Deltatax4Delta* cells because of a delay in bulk autophagy. Irs4p and Tax4p are also required for pexophagy but not for the cytoplasm-to-vacuole pathway. In growing cells, Irs4p and Tax4p colocalized to few cytoplasmic puncta distinct from endosomes and Golgi compartments. In conditions inducing autophagy, Irs4p and Tax4p partially localized to the pre-autophagosomal structure (PAS) and are required to efficiently recruit to the PAS Atg17p, a factor modulating the autophagic response. We propose that Irs4p and Tax4p are two redundant modulators of the autophagic processes acting upstream from Atg17p, possibly in the signaling events leading to the activation of the autophagic machinery in response to starvation.

Slm1 and slm2 are novel substrates of the calcineurin phosphatase required for heat stress-induced endocytosis of the yeast uracil permease.

The Ca2+/calmodulin-dependent phosphatase calcineurin promotes yeast survival during environmental stress. We identified Slm1 and Slm2 as calcineurin substrates required for sphingolipid-dependent processes. Slm1 and Slm2 bind to calcineurin via docking sites that are required for their dephosphorylation by calcineurin and are related to the PXIXIT motif identified in NFAT. In vivo, calcineurin mediates prolonged dephosphorylation of Slm1 and Slm2 during heat stress, and this response can be mimicked by exogenous addition of the sphingoid base phytosphingosine. Slm proteins also promote the growth of yeast cells in the presence of myriocin, an inhibitor of sphingolipid biosynthesis, and regulation of Slm proteins by calcineurin is required for their full activity under these conditions. During heat stress, sphingolipids signal turnover of the uracil permease, Fur4. In cells lacking Slm protein activity, stress-induced endocytosis of Fur4 is blocked, and Fur4 accumulates at the cell surface in a ubiquitinated form. Furthermore, cells expressing a version of Slm2 that cannot be dephosphorylated by calcineurin display an increased rate of Fur4 turnover during heat stress. Thus, calcineurin may modulate sphingolipid-dependent events through regulation of Slm1 and Slm2. These findings, in combination with previous work identifying Slm1 and Slm2 as targets of Mss4/phosphatidylinositol 4,5-bisphosphate and TORC2 signaling, suggest that Slm proteins integrate information from a variety of signaling pathways to coordinate the cellular response to heat stress.

The GTPase-activating enzyme Gyp1p is required for recycling of internalized membrane material by inactivation of the Rab/Ypt GTPase Ypt1p.

Rab/Ypt GTPases are key regulators of membrane trafficking and together with SNARE proteins mediate selective fusion of vesicles with target compartments. A family of GTPase-activating enzymes (GAPs) specific for Rab/Ypt GTPases has been discovered, but little is known about their function and substrate specificity in vivo. Here we show that the GAP activity of Gyp1p, a yeast member of this family, is specifically required for recycling of the SNARE Snc1p and the membrane dye FM4-64, implying that inactivation of a Rab/Ypt GTPase may be necessary for recycling of membrane material. Interestingly, recycling of GFP-Snc1p in gyp1 Delta cells is partially restored by reducing the activity of Ypt1p. Moreover, GFP-Snc1p accumulated intracellularly in wild-type cells expressing a GTP-locked, mutant form of Ypt1p (Ypt1p-Q67L), suggesting that GTP hydrolysis of Ypt1p is essential for recycling. Ypt6p is known to be required for the fusion of recycling vesicles to the late Golgi compartment. Interestingly, the deletions of GYP1 and YPT6 were synthetic lethal, raising the possibility that at least two distinct pathways are involved in recycling of membrane material.

Antagonistic roles of ESCRT and Vps class C/HOPS complexes in the recycling of yeast membrane proteins.

In Saccharomyces cerevisiae, deficiencies in the ESCRT machinery trigger the mistargeting of endocytic and biosynthetic ubiquitinated cargoes to the limiting membrane of the vacuole. Surprisingly, impairment of this machinery also leads to the accumulation of various receptors and transporters at the plasma membrane in both yeast and higher eukaryotes. Using the well-characterized yeast endocytic cargo uracil permease (Fur4p), we show here that the apparent stabilization of the permease at the plasma membrane in ESCRT mutants results from an efficient recycling of the protein. Whereas several proteins as well as internalized dyes are known to be recycled in yeast, little is known about the machinery and molecular mechanisms involved. The SNARE protein Snc1p is the only cargo for which the recycling pathway is well characterized. Unlike Snc1p, endocytosed Fur4p did not pass through the Golgi apparatus en route to the plasma membrane. Although ubiquitination of Fur4p is required for its internalization, deubiquitination is not required for its recycling. In an attempt to identify actors in this new recycling pathway, we found an unexpected phenotype associated with loss of function of the Vps class C complex: cells defective for this complex are impaired for recycling of Fur4p, Snc1p, and the lipophilic dye FM4-64. Genetic analyses indicated that these phenotypes were due to the functioning of the Vps class C complex in trafficking both to and from the late endosomal compartment.

Opposite roles of the F-box protein Rcy1p and the GTPase-activating protein Gyp2p during recycling of internalized proteins in yeast.

The F-box protein Rcy1p is part of a non-SCF (Skp1p-cullin-F-box protein) complex involved in recycling of internalized material. Like rcy1Delta, cells lacking the Rab-GTPase Ypt6p or its heterodimeric GEFs Rgp1p and Ric1p are unable to recycle the v-SNARE Snc1p. Here we provide genetic evidence suggesting that Rcy1p is a positive regulator of Ypt6p. Deletion of the GAP Gyp2p restores recycling in rcy1Delta, while overexpression of an active form of Ypt6p partially suppresses the recycling defect of rcy1Delta cells. Conversely, overexpression of Gyp2p in wild-type cells interferes with recycling of GFP-Snc1p, and the cells accumulate membrane structures as evidenced by electron microscopy. Gyp2p-GFP is distributed throughout the cytoplasm and accumulates in punctate structures, which concentrate in an actin-dependent manner at sites of polarized growth. Taken together, our results suggest that the F-box protein Rcy1p may activate the Ypt6p GTPase module during recycling.

Proteasome- and SCF-dependent degradation of yeast adenine deaminase upon transition from proliferation to quiescence requires a new F-box protein named Saf1p.

In response to nutrient limitation, Saccharomyces cerevisiae cells enter into a non-proliferating state termed quiescence. This transition is associated with profound changes in gene expression patterns. The adenine deaminase encoding gene AAH1 is among the most precociously and tightly downregulated gene upon entry into quiescence. We show that AAH1 downregulation is not specifically due to glucose exhaustion but is a more general response to nutrient limitation. We also found that Aah1p level is tightly correlated to RAS activity indicating thus an important role for the protein kinase A pathway in this regulation process. We have isolated three deletion mutants, srb10, srb11 and saf1 (ybr280c) affecting AAH1 expression during post-diauxic growth and in early stationary phase. We show that the Srb10p cyclin-dependent kinase and its cyclin, Srb11p, regulate AAH1 expression at the transcriptional level. By contrast, Saf1p, a previously uncharacterized F-box protein, acts at a post-transcriptional level by promoting degradation of Aah1p. This post-transcriptional regulation is abolished by mutations affecting the proteasome or constant subunits of the SCF (Skp1-Cullin-F-box) complex. We propose that Saf1p targets Aah1p for proteasome-dependent degradation upon entry into quiescence. This work provides the first direct evidence for active degradation of proteins in quiescent yeast cells.

Yeast functional analysis: identification of two essential genes involved in ER to Golgi trafficking.

We screened for genes potentially involved in the secretory and vacuolar pathways a collection of 61 yeast strains, each bearing an essential orphan gene regulated by the tetO7-CYC1 promoter that can be down-regulated by doxycycline. After down-regulating the expression of these genes, we performed systematic Western blot analysis for markers of the secretory and vacuolar pathways that undergo post-translational modifications in their intracellular trafficking. Accumulation of protein precursors, revealed by Western immunoblot analysis, indicates defects in the secretory pathway or in associated biochemical modifications. After screening the whole collection, we identified two genes involved in ER to Golgi trafficking: RER2, a cis-prenyl transferase, and USE1, the function of which was unknown. We demonstrated that repression of USE1 also leads to BiP secretion, and therefore likely affects retrograde, in addition to anterograde, ER to Golgi trafficking. The collection also includes two essential genes involved in intracellular trafficking that were conveniently repressed without resulting growth or trafficking defects.