Complementary α-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids.

How cells adjust nutrient transport across their membranes is incompletely understood. Previously, we have shown that S. cerevisiae broadly re-configures the nutrient transporters at the plasma membrane in response to amino acid availability, through endocytosis of sugar- and amino acid transporters (AATs) (Müller et al., 2015). A genome-wide screen now revealed that the selective endocytosis of four AATs during starvation required the α-arrestin family protein Art2/Ecm21, an adaptor for the ubiquitin ligase Rsp5, and its induction through the general amino acid control pathway. Art2 uses a basic patch to recognize C-terminal acidic sorting motifs in AATs and thereby instructs Rsp5 to ubiquitinate proximal lysine residues. When amino acids are in excess, Rsp5 instead uses TORC1-activated Art1 to detect N-terminal acidic sorting motifs within the same AATs, which initiates exclusive substrate-induced endocytosis. Thus, amino acid excess or starvation activate complementary α-arrestin-Rsp5-complexes to control selective endocytosis and adapt nutrient acquisition.

Regulation of Rab5 isoforms by transcriptional and post-transcriptional mechanisms in yeast.

Rab5 GTPases are master regulators of early endosome biogenesis and transport. The genome of Saccharomyces cerevisiae encodes three Rab5 proteins: Vps21, the major isoform, Ypt52 and Ypt53. Here, we show that Vps21 is the most abundant Rab5 protein and Ypt53 is the least abundant. In stressed cells, Ypt53 levels increase but never exceed that of Vps21. Its induction requires the transcription factors Crz1 and Gis1. In growing cells, the expression of Ypt53 is suppressed by post-transcriptional mechanisms mediated by the untranslated regions of the YPT53 mRNA. Based on genetic experiments, these sequences appear to stimulate deadenylation, Pat1-activated decapping and Xrn1-mediated mRNA degradation. Once this regulation is bypassed, Ypt53 protein levels surpass Vps21, and Ypt53 is sufficient to maintain endosomal function and cell growth.

The coordinated action of the MVB pathway and autophagy ensures cell survival during starvation.

The degradation and recycling of cellular components is essential for cell growth and survival. Here we show how selective and non-selective lysosomal protein degradation pathways cooperate to ensure cell survival upon nutrient limitation. A quantitative analysis of starvation-induced proteome remodeling in yeast reveals comprehensive changes already in the first three hours. In this period, many different integral plasma membrane proteins undergo endocytosis and degradation in vacuoles via the multivesicular body (MVB) pathway. Their degradation becomes essential to maintain critical amino acids levels that uphold protein synthesis early during starvation. This promotes cellular adaptation, including the de novo synthesis of vacuolar hydrolases to boost the vacuolar catabolic activity. This order of events primes vacuoles for the efficient degradation of bulk cytoplasm via autophagy. Hence, a catabolic cascade including the coordinated action of the MVB pathway and autophagy is essential to enter quiescence to survive extended periods of nutrient limitation.

Stability of the endosomal scaffold protein LAMTOR3 depends on heterodimer assembly and proteasomal degradation.

LAMTOR3 (MP1) and LAMTOR2 (p14) form a heterodimer as part of the larger Ragulator complex that is required for MAPK and mTOR1 signaling from late endosomes/lysosomes. Here, we show that loss of LAMTOR2 (p14) results in an unstable cytosolic monomeric pool of LAMTOR3 (MP1). Monomeric cytoplasmic LAMTOR3 is rapidly degraded in a proteasome-dependent but lysosome-independent manner. Mutational analyses indicated that the turnover of the protein is dependent on ubiquitination of several lysine residues. Similarly, other Ragulator subunits, LAMTOR1 (p18), LAMTOR4 (c7orf59), and LAMTOR5 (HBXIP), are degraded as well upon the loss of LAMTOR2. Thus the assembly of the Ragulator complex is monitored by cellular quality control systems, most likely to prevent aberrant signaling at the convergence of mTOR and MAPK caused by a defective Ragulator complex.

Assembly of the PtdIns 4-kinase Stt4 complex at the plasma membrane requires Ypp1 and Efr3.

The phosphoinositide phosphatidylinositol 4-phosphate (PtdIns4P) is an essential signaling lipid that regulates secretion and polarization of the actin cytoskeleton. In Saccharomyces cerevisiae, the PtdIns 4-kinase Stt4 catalyzes the synthesis of PtdIns4P at the plasma membrane (PM). In this paper, we identify and characterize two novel regulatory components of the Stt4 kinase complex, Ypp1 and Efr3. The essential gene YPP1 encodes a conserved protein that colocalizes with Stt4 at cortical punctate structures and regulates the stability of this lipid kinase. Accordingly, Ypp1 interacts with distinct regions on Stt4 that are necessary for the assembly and recruitment of multiple copies of the kinase into phosphoinositide kinase (PIK) patches. We identify the membrane protein Efr3 as an additional component of Stt4 PIK patches. Efr3 is essential for assembly of both Ypp1 and Stt4 at PIK patches. We conclude that Ypp1 and Efr3 are required for the formation and architecture of Stt4 PIK patches and ultimately PM-based PtdIns4P signaling.