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 homogenates from Dictyostelium discoideum cells fed colloidal iron, were lysed under hypoosmotic conditions, and the membrane-associated proteins were subjected to gel electrophoresis. Thirteen major membrane polypeptides, ranging in molecular weight from 25,000 to 100,000 were identified. The isoelectric points of these proteins ranged from below 3.8 to greater than 7.0. Most of these proteins were stripped from membranes exposed to a chaotropic agent, 3,5-diodo-2-hydroxybenzoic acid lithium salt, and were therefore classified as peripheral membrane proteins. Twenty five glycoprotein species were detected by lectin blot analysis; 19 were classified as integral membrane proteins, and were, in general, larger than 45 kDa and negatively charged due in part to the presence of mannose 6-sulfate. Western blot analysis also demonstrated that a Rab 4-like GTPase, a Rab 7-like GTPase, and at least three subunits of the vacuolar ATPase were associated with the lysosomal membrane; the ATPase subunits appeared to be major proteins in lysosomal membranes. 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 ATPase subunit of approximately 41 kDa. The D. discoideum DVA41 gene was apparently a single copy gene, expressed at constant levels during growth and development.
Temesvari
L
L
Department of Microbiology and Immunology, LSU Medical Center, Shreveport 71130.
Rodriguez-Paris
J
J
Bush
J
J
Steck
T L
TL
Cardelli
J
J
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002458:Cell Fractionation / Q000379:methods; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development; D015180:Electrophoresis, Gel, Two-Dimensional; D019204:GTP-Binding Proteins / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis; D007425:Intracellular Membranes / Q000737:chemistry*; D008247:Lysosomes / Q000737:chemistry* / Q000201:enzymology; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D025262:Vacuolar Proton-Translocating ATPases
D006023:Glycoproteins; D008565:Membrane Proteins; D015800:Protozoan Proteins; D000251:Adenosine Triphosphatases; D019204:GTP-Binding Proteins; D025262:Vacuolar Proton-Translocating ATPases; C089945:DVA41 protein, Dictyostelium discoideum