CHARACTERIZATION OF LYSOSOMAL MEMBRANE-PROTEINS OF DICTYOSTELIUM-DISCOIDEUM - A COMPLEX POPULATION OF ACIDIC INTEGRAL MEMBRANE-GLYCOPROTEINS, RAB GTP-BINDING PROTEINS AND VACUOLAR ATPASE SUBUNITS

Highly purified lysosomes, prepared by magnetic fractionation of homog enates from Dictyostelium discoideum cells fed colloidal iron, were ly sed under hypoosmotic conditions, and the membrane associated proteins were subjected to gel electrophoresis. Thirteen major membrane polype ptides, ranging in molecular weight from 25,000 to 100,000 were identi fied. The isoelectric points of these proteins ranged from below 3.8 t o greater than 7.0. Most of these proteins were stripped from membrane s exposed to a chaotropic agent, 3,5-diiodo-2-hydroxybenzoic acid lith ium salt, and were therefore classified as peripheral membrane protein s. Twenty five glycoprotein species were detected by lectin blot analy sis; 19 were classified as integral membrane proteins, and were, in ge neral,larger than 45 kDa and negatively charged due in part to the pre sence of mannose B-sulfate. Western blot analysis also demonstrated th at a Rab 4-like GTPase, a Rab 7-like GTPase, and at least three subuni ts of the vacuolar ATPase were associated with the lysosomal membrane; the ATPase subunits appeared to be major proteins in lysosomal membra nes. Finally, based on N-terminal sequence analysis of a major 41-kDa lysosome-associated membrane protein, we cloned a cDNA that encodes a protein (DVA41) highly homologous to a yeast and a bovine vacuolar ATP ase subunit of approximately 41 kDa. The D. discoideum DVA41 gene was apparently a single copy gene, expressed at constant levels during gro wth and development.