The human phosphatidylinositol phosphatase SAC1 interacts with the coatomer I complex.
The Saccharomyces cerevisiae SAC1 gene encodes an integral membrane protein of the endoplasmic reticulum (ER) and the Golgi apparatus. Yeast SAC1 mutants display a wide array of phenotypes including inositol auxotrophy, cold sensitivity, secretory defects, disturbed ATP transport into the ER, or suppression of actin gene mutations. At present, it is not clear how these phenotypes relate to the finding that SAC1 displays polyphosphoinositide phosphatase activity. Moreover, it is still an open question whether SAC1 functions similarly in mammalian cells, since some phenotypes are yeast-specific. Potential protein interaction partners and, connected to that, possible regulatory circuits have not been described. Therefore, we have cloned human SAC1 (hSAC1), show that it behaves similar to ySac1p in terms of substrate specificity, demonstrate that the endogenous protein localizes to the ER and Golgi, and identify for the first time members of the coatomer I (COPI) complex as interaction partners of hSAC1. Mutation of a putative COPI interaction motif (KXKXX) at its C terminus abolishes interaction with COPI and causes accumulation of hSAC1 in the Golgi. In addition, we generated a catalytically inactive mutant, demonstrate that its lipid binding capacity is unaltered, and show that it accumulates in the Golgi, incapable of interacting with the COPI complex despite the presence of the KXKXX motif. These results open the possibility that the enzymatic function of hSAC1 provides a switch for accessibility of the COPI interaction motif.
Rohde
Holger M
HM
Boehringer Ingelheim Austria GmbH, Dr. Boehringer-Gasse 5-11, 1121 Vienna, Austria.
Cheong
Fei Ying
FY
Konrad
Gerlinde
G
Paiha
Karin
K
Mayinger
Peter
P
Boehmelt
Guido
G
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002384:Catalysis; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D020756:Coatomer Protein / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005816:Genetic Complementation Test; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D050356:Lipid Metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology*; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D012568:Schizosaccharomyces / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D020756:Coatomer Protein; D018076:DNA, Complementary; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010455:Peptides; C416669:Sac1 protein, mammalian; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; C081864:SAC1 protein, S cerevisiae; C089504:polyphosphoinositide phosphatase; D010744:Phosphoric Monoester Hydrolases