Our genome-wide analysis for suppressors of sec14ts
temperature-sensitivity has led to the identification of a previously unknown role for Sec14, ensuring proper trafficking and localization of lipid raft resident proteins. We suggest this is a major essential function of the phospholipid transfer protein Sec14. Herein, we described that the sec14ts
encoded protein, Sec14G266D
, was normally present in cells at a lower level than the wild type Sec14 protein, and increasing Sec14G266D
level by inhibition of proteasome function or increased dose of the Sec14G266D
protein itself, relieved the growth defect of sec14ts
cells. Fus-Mid-GFP and Pma1 localization were defective in sec14ts
cells and restored upon increased expression of Sec14G266D
, whereas none of the previously identified vesicular trafficking defects associated with loss of Sec14 function were restored, and membrane accumulation still occurred as determined by electron microscopy. Previous work had observed that membrane accumulation was still present in ‘sec14
bypass suppressor’ cells that had an inactivated SEC14
gene in combination with inactivating genes for the CDP-choline pathway for PC synthesis, a condition that restored growth to cells lacking Sec14 function 
. Membrane accumulation and vesicular trafficking defects are clearly present in cells with reduced Sec14 function, but these do not appear to be the major contributing phenotypes to reduced growth as our findings indicate there is no correlation between growth restoration, membrane accumulation, and defects in the vesicular trafficking pathways previously identified as defective sec14ts
cells. Instead, an inability to traffic and localize lipid raft resident proteins appears to be a major function of Sec14.
Three main classes of plasma membrane domains, collectively referred to as lipid rafts, have so far been identified in yeast 
. The (i) membrane compartment containing Can1 (MCC, also referred to as eisosomes) are enriched in transporters and are thought to have high sterol content, (ii) the membrane compartment containing target of rapamycin kinase complex 2 (MCT) domains have a yet unknown origin, and (iii) the membrane compartment containing Pma1 (MCP) domains. Recently, a fourth highly ordered membrane domain was described that was sterol free and sphingolipid enriched that may play a role in organization of GPI-anchored proteins 
. Pma1 mislocalization was noted in this study, it will be of interest to assess if Sec14 function also regulates assembly and function of the other types of lipid raft domains.
In this study, we also determined that a reporter of protein assembly into lipid rafts, Fus-Mid-GFP, was compromised in sec14ts
cells and this was partially restored by increased Sec14G266D
levels. The Fus-Mid-GFP protein is selectively sorted into sterol and sphingolipid rich domains at the trans
-Golgi, with this sorting being required for Fus-Mid-GFP trafficking from the Golgi to the plasma membrane, and defects in either sterol or sphingolipid synthesis compromise Fus-Mid-GFP trafficking to the plasma membrane 
. Prior to Pma1 localization to MCP lipid rafts at the plasma membrane, Pma1 associates with lipid rafts that are forming in the Golgi. Defects in sphingolipid synthesis result in an inability to sort Pma1 into lipid rafts at the Golgi resulting in defective Pma1 trafficking from this organelle 
. Pma1 can also be mislocalized subsequent to delivery to the plasma membrane due to alterations in its ability to maintain association with MCP lipid rafts once at the plasma membrane 
. Based on the results from the work presented here, Sec14 may mediate sorting of proteins that are dependent on sphingolipid synthesis for partitioning into lipid rafts at the Golgi for their delivery to the plasma membrane.
Other studies recently linked Sec14 function with sphingolipid metabolism. One study reported that in sec14ts
cells there was a 3–4 fold increase in ceramide mass and changes in the level/metabolism of complex sphingolipids 
. Another link between Sec14 and sphingolipid metabolism has recently emerged as inactivation of the SAC1
gene encoding the PI-4P phosphatase can bypass the essential function of Sec14, and the PI produced by Sac1 has been demonstrated to be preferentially used as substrate by Aur1 to convert ceramides into complex sphingolipids 
. A second gene, KES1
, can also bypass the essential function of Sec14 and has recently been determined to be an activator of Sac1 PI-4P phosphatase activity 
. It is clear that sphingolipid metabolism and Sec14 function are linked through both genetic interactions as well as through regulation of sphingolipid levels themselves. Our data imply that regulation of lipid raft protein trafficking and assembly is a node where Sec14 regulation of lipid metabolism and cell function converge.