Lag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum. lag1⌬ lac1⌬ double mutants in Saccharomyces cerevisiae lack an acyl-CoA-dependent ceramide synthase and are either very sick or nonviable, depending on the genetic background. LAG1 and LAC1 are members of a large eukaryotic gene family that shares the Lag1 motif, and some members of this family additionally contain a DNA-binding HOX homeodomain. Here we show that several human LAG1 homologues can rescue the viability of lag1⌬ lac1⌬ yeast cells and restore acyl-CoA-dependent ceramide and sphingolipid biosynthesis. When tested in a microsomal assay, Lac1p and Lag1p had a strong preference for C26:0-CoA over C24:0-CoA, C20-CoA, and C16-CoA, whereas some human homologues preferred C24:0-CoA and CoA derivatives with shorter fatty acids. This suggests that LAG1 proteins are related to substrate recognition and to the catalytic activity of ceramide synthase enzymes. CLN8, another human LAG1 homologue implicated in ceroid lipofuscinosis, could not restore viability to lag1⌬ lac1⌬ yeast mutants.The only structural sphingolipids of Saccharomyces cerevisiae are the inositol phosphorylceramides (IPCs), 1 mannosylIPCs (MIPCs), and inositol phosphoryl-MIPC, which together represent a significant fraction of membrane lipids, especially in the plasma membrane (1-3). Recent progress has resulted in the identification of yeast genes involved in all enzymatic steps that are required for their biosynthesis (4). The intermediates in sphingolipid synthesis, dihydrosphingosine (DHS), phytosphingosine (PHS), and their 1-phosphorylated derivatives, as well as free ceramides, have been proposed to act as signal transduction molecules governing heat stress responses, endocytosis, glycosyl phosphatidylinositol protein transport, ubiquitin-dependent degradation of membrane channels, and progression through G 1 (for review, see Ref. 4).A key role in the sphingolipid pathway is played by ceramide synthase, as it not only catalyzes an essential biosynthetic reaction, but also influences the levels of long chain bases and ceramides, which have signaling function. The simultaneous deletion of LAG1 and its close homologue LAC1 eliminates all detectable acyl-CoA-dependent ceramide biosynthesis in yeast microsomes (5, 6). Moreover, lag1⌬ lac1⌬ cells have a drastically reduced amount of normal ceramides and IPCs, but exhibit a marked accumulation of free DHS and a compensatory increase of C26:0 fatty acids, which seem to be used for making a new form of phosphatidylinositol (PI) that we call PIЈ (5, 6). Whereas the single deletion of LAG1 or LAC1 had no abnormal growth phenotype, the concomitant deletion of LAG1 and LAC1 caused osmotic fragility, calcofluor white hypersensitivity, and a significant decrease of the growth rate in the genetic background of W303 cells, and the same double mutation was lethal in the background of YPK9 cells (5,7,8). Lethality of YPK9 lag1⌬lac1⌬ (herein named YPK9.2⌬) can be overcome by overexpression of LAG1 homologues from man (LA...

SummaryAll mature Saccharomyces cerevisiae sphingolipids comprise inositolphosphorylceramides containing C26:0 or C24:0 fatty acids and either phytosphingosine or dihydrosphingosine. Here we analysed the lipid profile of lag1D lac1D mutants lacking acyl-CoAdependent ceramide synthesis, which require the reverse ceramidase activity of overexpressed Ydc1p for sphingolipid biosynthesis and viability. These cells, termed 2D.YDC1, make sphingolipids containing exclusively dihydrosphingosine and an abnormally wide spectrum of fatty acids with between 18 and 26 carbon atoms. Like wild-type cells, 2D.YDC1 cells stop growing when exposed to Aureobasidin A (AbA), an inhibitor of the inositolphosphorylceramide synthase AUR1, yet their ceramide levels remain very low. This finding argues against a current hypothesis saying that yeast cells do not require inositolphosphorylceramides and die in the presence of AbA only because ceramides build up to toxic concentrations. Moreover, W303lag1D lac1D ypc1D ydc1D cells, reported to be AbA resistant, stop growing on AbA after a certain number of cell divisions, most likely because AbA blocks the biosynthesis of anomalous inositolphosphorylsphingosides. Thus, data argue that inositolphosphorylceramides of yeast, the equivalent of mammalian sphingomyelins, are essential for growth. Data also clearly confirm that wild-type strains, when exposed to AbA, immediately stop growing because of ceramide intoxication, long before inositolphosphorylceramide levels become subcritical.

CWH8/YGR036c of Saccharomyces cerevisiae has been identified as a dolichylpyrophosphate (Dol-PP) phosphatase that removes a phosphate from the Dol-PP generated by the oligosaccharyltransferase (OST), while it adds N-glycans to nascent glycoproteins in the endoplasmic reticulum (ER). Lack of CWH8 was proposed to interrupt the so called dolichol (Dol) cycle by trapping Dol in the form of Dol-PP in the ER lumen. Indeed, cwh8Delta mutants display a severe deficiency in N-glycosylation. We find that cwh8Delta mutants have strongly reduced levels of inositolphosphorylceramide (IPC), whereas its derivative, mannosyl-(inositol-P)2-ceramide (M(IP)2C) is not affected. Microsomes of cwh8Delta contain normal ceramide synthase and IPC synthesis activities. Within a large panel of mutants affecting Dol dependent pathways such as N- or O-glycosylation, or glycosylphosphatidyl inositol (GPI)-anchoring, only the mutants having a deficiency of N-glycan addition show the defect in IPC biosynthesis. By mutating genes required for the addition of N-glycans or by treating cells with tunicamycin (Tm) one can similarly reduce the steady state level of IPC and exactly reproduce the phenotype of cwh8Delta cells. Some potential mechanisms by which the lack of N-glycans could lead to the sphingolipid abnormality were further explored.

Receptor‐mediated endocytosis is a pivotal function of the renal proximal tubule (PT) to reabsorb proteins from the ultra filtrate. PT dysfunction occurring in nephrotic syndrome results in an impairment of this process. The regulation of endocytosis and signaling pathways involved remain unknown. We aimed to identify whether low (0.1mg/ml) vs. high albumin concentration (10mg/ml) affect endocytosis and to uncover responsible signaling pathways. OK‐cells incubated with 0.1mg/ml albumin significantly augmented receptor‐mediated endocytosis at 1h and 4h. In contrary, incubating OKC with 10mg/ml albumin significantly diminished endocytosis after 30 min till 4h. Analyses of the expression pattern and phosphorylation status of the kinases including PI3K, Akt, TORC1/‐2, WNK1/OSR1 revealed that the WNK1‐induced pathway shows strongest changes upon low vs. high albumin concentrations. Transient transfection of OKC using WNK1, KD‐K233M‐WNK1, OSR1 and KD‐D164A‐OSR1 strongly inhibited endocytosis to 2.8±1.1*; 6.7±1.0*; 2.8±0.5*; and 7.4±2.9*; *P<0.05, respectively, suggesting a regulation by protein‐protein interaction. In rat kidney, IHC of WNK1 and OSR1 demonstrated a strong expression in the BBM of PT, supporting a role in endocytosis. We conclude that WNK1 and OSR1 are the main kinases activated upon high protein concentrations leading to consequent impairment of receptor‐mediated endocytosis.