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Epithelial cell physiology critically depends on the asymmetric distribution of channels and transporters. However, the mechanisms targeting membrane proteins to the apical surface are still poorly understood. Here, we performed a visual forward genetic screen in the zebrafish intestine and identified mutants with defective apical targeting of membrane proteins. One of these mutants, affecting the vacuolar H+-ATPase gene atp6ap1b, revealed specific requirements for luminal acidification in apical, but not basolateral, membrane protein sorting and transport. Using a low temperature block assay combined with genetic and pharmacologic perturbation of luminal pH, we monitored transport of newly synthesized membrane proteins from the TGN to apical membrane in live zebrafish. We show that vacuolar H+-ATPase activity regulates sorting of O-glycosylated proteins at the TGN, as well as Rab8-dependent post-Golgi trafficking of different classes of apical membrane proteins. Thus, luminal acidification plays distinct and specific roles in apical membrane biogenesis., (© 2020 Levic et al.)

Recently synthesized proteins are sorted at the trans-Golgi network into specialized routes for exocytosis. Surprisingly little is known about the underlying molecular machinery. Here, we present a visual screen to search for proteins involved in cargo sorting and vesicle formation. We expressed a GFP-tagged plasma membrane protein in the yeast deletion library and identified mutants with altered marker localization. This screen revealed a requirement of several enzymes regulating the synthesis of sphingolipids and ergosterol in the correct and efficient delivery of the marker protein to the cell surface. Additionally, we identified mutants regulating the actin cytoskeleton (Rvs161p and Vrp1p), known membrane traffic regulators (Kes1p and Chs5p), and several unknown genes. This visual screening method can now be used for different cargo proteins to search in a genome-wide fashion for machinery involved in post-Golgi sorting.

Protein O mannosylation is a crucial protein modification in uni- and multicellular eukaryotes. In humans, a lack of O-mannosyl glycans causes congenital muscular dystrophies that are associated with brain abnormalities. In yeast, protein O mannosylation is vital; however, it is not known why impaired O mannosylation results in cell death. To address this question, we analyzed the conditionally lethal Saccharomyces cerevisiae protein O-mannosyltransferase pmt2 pmt4Delta mutant. We found that pmt2 pmt4Delta cells lyse as small-budded cells in the absence of osmotic stabilization and that treatment with mating pheromone causes pheromone-induced cell death. These phenotypes are partially suppressed by overexpression of upstream elements of the protein kinase C (PKC1) cell integrity pathway, suggesting that the PKC1 pathway is defective in pmt2 pmt4Delta mutants. Congruently, induction of Mpk1p/Slt2p tyrosine phosphorylation does not occur in pmt2 pmt4Delta mutants during exposure to mating pheromone or elevated temperature. Detailed analyses of the plasma membrane sensors of the PKC1 pathway revealed that Wsc1p, Wsc2p, and Mid2p are aberrantly processed in pmt mutants. Our data suggest that in yeast, O mannosylation increases the activity of Wsc1p, Wsc2p, and Mid2p by enhancing their stability. Reduced O mannosylation leads to incorrect proteolytic processing of these proteins, which in turn results in impaired activation of the PKC1 pathway and finally causes cell death in the absence of osmotic stabilization.

Exposure to mating pheromone in haploid Saccharomyces cerevisiae cells results in the arrest of the cell cycle, expression of mating-specific genes, and polarized growth toward the mating partner. Proteins involved in signaling, polarization, cell adhesion, and fusion are localized to the tip of the mating cell (shmoo) where fusion will eventually occur. The mechanisms ensuring the correct targeting and retention of these proteins are poorly understood. Here we show that in pheromone-treated cells, a reorganization of the plasma membrane involving lipid rafts results in the retention of proteins at the tip of the mating projection, segregated from the rest of the membrane. Sphingolipid and ergosterol biosynthetic mutants fail to polarize proteins to the tip of the shmoo and are deficient in mating. Our results show that membrane microdomain clustering at the mating projection is involved in the generation and maintenance of polarity during mating.