Lst1p and Exp1p act in parallel pathways to export the plasma membrane H⁺-ATPase from the ER in S. cerevisiae
Author(s)Kirtley, Michelle Crotwell, 1974-
Massachusetts Institute of Technology. Dept. of Biology.
Chris A. Kaiser.
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Efficient transport of proteins to the correct intracellular compartment is critical for maintaining the functional integrity of the cell. Proteins destined for export from the ER are sorted from ER resident proteins and packaged into vesicles coated with the COPII protein complex. To facilitate our study of the mechanisms of protein sorting, we have selected Pma1p, the plasma membrane H+ ATPase of S. cerevisiae, as a model cargo protein. We found that efficient trafficking of Pma1p to the cell surface requires Lst1p, one of two yeast homologs of the COPII component Sec24p. We initially isolated LSTJ as one of a series of genes whose mutant alleles are lethal in combination with mutant alleles of the COPII gene SEC13. Strains deleted for LSTJ exhibit phenotypes attributable to a defect in Pma1p localization, including sensitivity to growth on acidic medium (pH 3.0) and decreased proton pumping activity. Pma1p accumulates in the ER of Ist1A strains, while other cargo molecules such as invertase and CPY are transported with wildtype kinetics. Like Sec24p, Lst1p specifically binds the COPII component Sec23p. Thus, we propose that Lst1p is an alternative COPII component that selectively exports Pma1p from the ER. We isolated EXP1 (ER-export of Pma1p) as a low-copy suppressor of the lethality displayed by Ist1-] sec13-1 double mutants. Expression of EXP1 from a centromeric plasmid suppresses the sensitivity of IstlA strains to growth on acidic medium and restores plasma membrane localization of Pma1p. Unlike stlA strains, exp1 strains grow normally under acidic conditions.(cont.) However, stl A explA double mutants are inviable and display severe Pma1p-trafficking defects. EXP1 encodes a 17 KD Type III integral membrane that cofractionates with ER in wild type cells and with the Golgi in sec21-1 mutants, indicating that Exp1p normally cycles between the Golgi and ER. Bacterially purified Exp1p fusion proteins interact with Sec23/24p in vitro. We have identified specific deletions of the cytosolic domain of EXP1 that fail to complement the lethality of a 1st1Aexp1A double mutant and to bind the Sec23/24p subcomplex. We also demonstrated that these deletion mutants fail to cycle between the Golgi and the ER. Based on these findings, we propose that Exp1p acts to enhance the Sec24p-mediated export of Pma1p from the ER. The identification here of two parallel pathways for Pma1p export from the ER provides new insight into the molecular mechanisms driving the selective uptake of secretory cargo proteins into budding COPII vesicles.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004.Includes bibliographical references.
DepartmentMassachusetts Institute of Technology. Dept. of Biology.
Massachusetts Institute of Technology