Cdc50p, a Protein Required for Polarized Growth, Associates with the Drs2p P-Type ATPase Implicated in Phospholipid Translocation inSaccharomyces cerevisiae

Abstract

Cdc50p, a transmembrane protein localized to the late endosome, is required for polarized cell growth in yeast. Genetic studies suggest that CDC50 performs a function similar to DRS2, which encodes a P-type ATPase of the aminophospholipid translocase (APT) subfamily. At low temperatures, drs2Delta mutant cells exhibited depolarization of cortical actin patches and mislocalization of polarity regulators, such as Bni1p and Gic1p, in a manner similar to the cdc50Delta mutant. Both Cdc50p and Drs2p were localized to the trans-Golgi network and late endosome. Cdc50p was coimmunoprecipitated with Drs2p from membrane protein extracts. In cdc50Delta mutant cells, Drs2p resided on the endoplasmic reticulum (ER), whereas Cdc50p was found on the ER membrane in drs2Delta cells, suggesting that the association on the ER membrane is required for transport of the Cdc50p-Drs2p complex to the trans-Golgi network. Lem3/Ros3p, a homolog of Cdc50p, was coimmunoprecipitated with another APT, Dnf1p; Lem3p was required for exit of Dnf1p out of the ER. Both Cdc50p-Drs2p and Lem3p-Dnf1p were confined to the plasma membrane upon blockade of endocytosis, suggesting that these proteins cycle between the exocytic and endocytic pathways, likely performing redundant functions. Thus, phospholipid asymmetry plays an important role in the establishment of cell polarity; the Cdc50p/Lem3p family likely constitute potential subunits specific to unique P-type ATPases of the APT subfamily.

Mutational analysis of the Lem3p-Dnf1p putative phospholipid-translocating P-type ATPase reveals novel regulatory roles for Lem3p and a carboxyl-terminal region of Dnf1p independent of the phospholipid-translocating activity of Dnf1p in yeast

“…The preparation of crude membrane fractions was performed as described previously [13]. For immunoprecipitation, membrane fractions were prepared from 200 OD 600 of cells and were solubilized in 0.8 ml of immunoprecipitation buffer (10 mM Tris⋅HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, and 1% CHAPS) containing protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 mM benzamidine, and 1 mM phenylmetylsulfonylfluoride).…”

“…immunoprecipitates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Western blotting was performed as described previously [13].…”

“…The preparation of large unilamellar vesicles was performed as described previously [13]. Fluorescently labeled phospholipid internalization experiments were performed as described previously [13,14].…”

“…Cdc50p, a member of the conserved membrane-spanning protein family, was identified as a gene required for polarized cell growth [12]. Cdc50p and its homolog, Lem3p, were subsequently shown to associate with Drs2p and Dnf1p, respectively, and to be required for the exit of these P-type ATPases from the endoplasmic reticulum (ER) [13]. Thus, disruption of the LEM3 gene results in a marked decrease in the internalization of fluorescencelabeled analogs of PE and PC across the plasma membrane [13][14][15].…”

“…Cdc50p and its homolog, Lem3p, were subsequently shown to associate with Drs2p and Dnf1p, respectively, and to be required for the exit of these P-type ATPases from the endoplasmic reticulum (ER) [13]. Thus, disruption of the LEM3 gene results in a marked decrease in the internalization of fluorescencelabeled analogs of PE and PC across the plasma membrane [13][14][15]. The Lem3p-Dnf1p and Cdc50p-Drs2p complexes are functionally redundant, because simultaneous 4 loss of function of both complexes results in a synthetic growth defect.…”