Trans-Golgi Network and Endosome Dynamics Connect Ceramide Homeostasis with Regulation of the Unfolded Protein Response and TOR Signaling in Yeast

Abstract

Synthetic genetic array analyses identify powerful genetic interactions between a thermosensitive allele (sec14-1ts ) of the structural gene for the major yeast phosphatidylinositol transfer protein (SEC14) and a structural gene deletion allele (tlg2⌬) for the Tlg2 target membrane-soluble N-ethylmaleimide-sensitive factor attachment protein receptor. The data further demonstrate Sec14 is required for proper trans-Golgi network (TGN)/endosomal dynamics in yeast. Paradoxically, combinatorial depletion of Sec14 and Tlg2 activities elicits trafficking defects from the endoplasmic reticulum, and these defects are accompanied by compromise of the unfolded protein response (UPR). UPR failure occurs downstream of Hac1 mRNA splicing, and it is further accompanied by defects in TOR signaling. The data link TGN/endosomal dynamics with ceramide homeostasis, UPR activity, and TOR signaling in yeast, and they identify the Sit4 protein phosphatase as a primary conduit through which ceramides link to the UPR. We suggest combinatorial Sec14/Tlg2 dysfunction evokes inappropriate turnover of complex sphingolipids in endosomes. One result of this turnover is potentiation of ceramideactivated phosphatase-mediated down-regulation of the UPR. These results provide new insight into Sec14 function, and they emphasize the TGN/endosomal system as a central hub for homeostatic regulation in eukaryotes.

Tracing Putative Trafficking of the Glycolytic Enzyme Enolase via SNARE-Driven Unconventional Secretion

“…In addition to its involvement in the Cvt pathway, Tlg2p is a syntaxin-like t-SNARE that participates in vesicle fusion, endocytosis, Golgi complex-to-vacuole transport, endosomal protein sorting, and protein release from the endoplasmic reticulum (1,10,18,20,35,41). Mousley and colleagues previously showed that Tlg2p has a role in protein secretion in combination with Sec14p (35), and our results suggest that the participation of Tlg2p in the secretion of Eno2p is plausible. Therefore, we conclude that the secretory pathway of glycolytic enzymes is regulated in a different manner from the Cvt pathway.…”

Comparative Analysis of Transcriptome and Fitness Profiles Reveals General and Condition-Specific Cellular Functions Involved in Adaptation to Environmental Change in Saccharomyces cerevisiae

“…A constant (targeted) flux of secretory vesicles, and a balancing flux of endocytic vesicles to recover membrane material are required for cellular remodeling as well as for cell growth (Araujo-Bazan et al, 2008;Higuchi et al, 2009;Kohli et al, 2008;Zonia and Munnik, 2008), also in Saccharomyces (Ayscough, 2005;Moreira et al, 2009;Walther et al, 2006). This appears central for maintenance of cellular homeostasis (Mousley et al, 2008). The high variability of the fitness effects of these mutant groups was not mimicked by a high variability of their transcription.…”

A Chemical Genetic Screen for Modulators of Exocytic Transport Identifies Inhibitors of a Transport Mechanism Linked to GTR2 Function

“…It is also possible that Avl9 and Gtr proteins have a role in regulating plasma membrane homeostasis, or an Avl9-and Gtr-dependent sorting or transport process depends on optimized lipid composition. There is much evidence that regulation lipid composition is critical for cargo sorting and transport carrier formation in the late secretory pathway (21,23,24,30,41).…”

“…In our samples, we did not observe the very large rings seen in the arf1⌬ mutant. However, the arf1⌬ and sec14-ts mutants have defects in transport from both Golgi and endosomes (15,23), and the invertase-transporting exocytic pathway likely transits endosomes (17), so our group A compounds could perturb exit from either or both the Golgi and endosomes. Wild-type cells had a similar but less abundant membraneaccumulation phenotype when grown in the presence of KU#7 (Fig.…”