CHARACTERIZATION OF LYSOSOMAL MEMBRANE-PROTEINS OF DICTYOSTELIUM-DISCOIDEUM - A COMPLEX POPULATION OF ACIDIC INTEGRAL MEMBRANE-GLYCOPROTEINS, RAB GTP-BINDING PROTEINS AND VACUOLAR ATPASE SUBUNITS
L. Temesvari et al., CHARACTERIZATION OF LYSOSOMAL MEMBRANE-PROTEINS OF DICTYOSTELIUM-DISCOIDEUM - A COMPLEX POPULATION OF ACIDIC INTEGRAL MEMBRANE-GLYCOPROTEINS, RAB GTP-BINDING PROTEINS AND VACUOLAR ATPASE SUBUNITS, The Journal of biological chemistry, 269(41), 1994, pp. 25719-25727
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.