Altering Sphingolipid Metabolism in Saccharomyces cerevisiae Cells Lacking the Amphiphysin Ortholog Rvs161 Reinitiates Sugar Transporter Endocytosis

Abstract

Amphiphysins are proteins thought to be involved in synaptic vesicle endocytosis. Amphiphysins share a common BAR domain, which can sense and/or bend membranes, and this function is believed to be essential for endocytosis. Saccharomyces cerevisiae cells lacking the amphiphysin ortholog Rvs161 are inviable when starved for glucose. Altering sphingolipid levels in rvs161 cells remediates this defect, but how lipid changes suppress remains to be elucidated. Here, we show that the sugar starvation-induced death of rvs161 cells extends to other fermentable sugar carbon sources, and the loss of sphingolipid metabolism suppresses these defects. In all cases, rvs161 cells respond to the starvation signal, elicit the appropriate transcriptional response, and properly localize the requisite sugar transporter(s). However, Rvs161 is required for transporter endocytosis. rvs161 cells accumulate transporters at the plasma membrane under conditions normally resulting in their endocytosis and degradation. Transporter endocytosis requires the endocytosis (endo) domain of Rvs161. Altering sphingolipid metabolism by deleting the very-long-chain fatty acid elongase SUR4 reinitiates transporter endocytosis in rvs161 and rvs161 endo ؊ cells. The sphingolipid-dependent reinitiation of endocytosis requires the ubiquitin-regulating factors Doa1, Doa4, and Rsp5. In the case of Doa1, the phospholipase A 2 family ubiquitin binding motif is dispensable. Moreover, the conserved AAA-ATPase Cdc48 and its accessory proteins Shp1 and Ufd1 are required. Finally, rvs161 cells accumulate monoubiquitin, and this defect is remediated by the loss of SUR4. These results show that defects in sphingolipid metabolism result in the reinitiation of ubiquitin-dependent sugar transporter endocytosis and suggest that this event is necessary for suppressing the nutrient starvation-induced death of rvs161 cells.

Dissecting BAR Domain Function in the Yeast Amphiphysins Rvs161 and Rvs167 during Endocytosis

“…Sphingolipids are required for endocytosis (Zanolari et al , 2000), and previous work has suggested their functional association with Rvs proteins (Desfarges et al , 1993; Balguerie et al , 2002; Germann et al , 2005; McCourt et al , 2009; Morgan et al , 2009). To further examine the functional relationship of Rvs proteins with sphingolipids, we asked whether changes in levels of complex sphingolipids affected either the fitness of BAR domain mutants or Rvs167-GFP localization.…”

Sphingosine and Sphingosine Kinase 1 Involvement in Endocytic Membrane Trafficking

“…Endophilins recruit dynamin and deform membranes and thus have essential roles in membrane curvature sensing and remodeling (8). Mutation of several genes in sphingolipid biosynthesis can also suppress endocytic defects resulting from deletion of RVS genes (6), and aberrant regulation of membrane sphingolipids interfered with their functions (7). A plasma membrane quantitative genetic interaction map further validated the links of eisosomes, protein-based structures that generate invaginations at the plasma membrane, with sphingolipid metabolism and endosomal trafficking (9).…”

“…Although the balance between cholesterol and sphingolipids within the plasma membrane has long been implicated in endocytic membrane trafficking (4,5), in contrast to cholesterol functions, little is still known about the roles of sphingolipids. However, genetic studies in yeast have suggested a functional link between sphingolipid metabolism and the endophilin homologs RVS161 and RVS167, which are Bin/Amphiphysin/Rvs (N-BAR) 3 domain-containing endocytic proteins (6,7). Endophilins recruit dynamin and deform membranes and thus have essential roles in membrane curvature sensing and remodeling (8).…”

Coupling between endocytosis and sphingosine kinase 1 recruitment

“…RVS161 and RVS167, and their metazoan homologues amphiphysin and endophilin, coordinate constriction of endocytic necks with other events required for the endocytic reaction 17 – 21 . Forward genetic screens for suppressors of rvs161 or rvs167 mutants identified mutations in enzymes of sphingolipid metabolism 22 , 23 . Subsequently, large-scale epistatic miniarray profile (E-MAP) studies extended these results by identifying additional enzymes of both sphingolipid and ergosterol metabolism that genetically interact with N-BAR proteins 24 .…”