The Human Phosphatidylinositol Phosphatase SAC1 Interacts with the Coatomer I Complex

Abstract

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.

The Sac1 Phosphoinositide Phosphatase Regulates Golgi Membrane Morphology and Mitotic Spindle Organization in Mammals

“…Under those conditions, mSac1-GFP colocalized with an ER marker (calnexin), a cis-Golgi marker (GM130), and a TGN marker (TGN46; Figure 3A). This pattern is in agreement with the distribution of endogenous Sac1 in mammalian cells (Nemoto et al, 2000;Rohde et al, 2003). mSac1-GFP showed a higher degree of colocalization with the cis-Golgi marker GM130 than with the TGN marker TGN46 ( Figure 3A).…”

“…Mammalian Sac1 harbors a C-terminal 583 KEKID 587 COP1-binding motif that promotes retrograde trafficking of this enzyme from Golgi compartments back to the ER. Inactivation of this motif by converting the K residues to A results in Sac1 accumulation in the Golgi system (Rohde et al, 2003). Expression of mSac1 AEAID -GFP, a mutant mSac1 for which the 583 KEKID 587 COP1-binding motif is com- Figure 9.…”

“…Mammalian Sac1 is an integral membrane protein with an anchor-sequence arrangement similar to that of ySac1p (Supplemental Figure S1, A and B), and it primarily resides in ER membranes (Nemoto et al, 2000). ER retention of the mammalian enzyme is controlled by a COP1-Sac1 binding interaction that captures enzyme in the Golgi system and recycles it back to the ER in COP1 vesicles (Rohde et al, 2003). Herein, we report the consequences of loss of Sac1 function in mice and in human cell models.…”

rSac3, a novel Sac domain phosphoinositide phosphatase, promotes neurite outgrowth in PC12 cells

“…The other subfamily of Sac domain-containing PIPPases includes two yeast proteins, Sac1p [21] and Fig4p [22], as well as several mammalian counterparts, hSac1 [23], hSac2 [24] and a yet-to-be-characterized putative protein KIAA0274. Yeast Sac1p was cloned as a suppressor of actin mutations [21].…”

A P4-ATPase Protein Interaction Network Reveals a Link between Aminophospholipid Transport and Phosphoinositide Metabolism

“…40 An association between Drs2p and Sec26p in the Golgi may account for this interaction. COPI subunits have been shown to physically interact with Sac1p, 41 suggesting that Sec26p might associate with Drs2p in a ternary complex with Sac1p. Tcb3p is a member of the tricalbin protein family.…”