Characterization of the interaction between p100, a novel G-protein-related protein, and rat liver endosomes.
p100 is a recently identified 100 kDa protein which shares a putative receptor-binding sequence with the signal transducing G-proteins Gt and Gi. In liver, p100 immunoreactivity is distributed between the cytosolic and the microsomal fractions [Traub, Evans & Sagi-Eisenberg (1990) Biochem. J. 272, 453-458; Udrisar & Rodbell (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6321-6325]. More specifically, we have localized the membrane-associated form of p100 to an endosomal subfraction of rat liver microsomes. In this study we have investigated the nature of the interaction between p100 and microsomal membranes. p100 was located on the cytoplasmic surface of the microsomal vesicles, and could be released by treatment with 0.5 M-NaCl or 0.5 M-Tris/HCl, pH 7.0. However, p100 was not released by non-ionic detergents, such as Triton X-100. Binding of p100 to the membrane was reversible, as both membrane-released and cytosolic p100 could re-bind stripped (Tris-washed) microsomes. Soluble p100 could not, however, bind to untreated microsomes. Binding to stripped microsomes approached saturation and was inhibited by up to 60% by either heat treatment or mild trypsin treatment of the vesicles. This implies that the interaction between p100 and the microsomal vesicles involves the direct binding of p100 to vesicular proteins. This binding was regulated by both adenine and guanine nucleotides. As p100 contains a region similar to the C-terminal decapeptide of alpha i, (the alpha-subunit of Gi) and has a localization that is restricted to an endosomal subfraction, we propose that cytosolic p100 may bind to cytoplasmically exposed domains of internalized receptors. Thus, like the adaptins, p100 may be involved in the process of sorting and receptor trafficking through the endosomal compartment of the cells.
Traub
L M
LM
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Shai
E
E
Sagi-Eisenberg
R
R
D000818:Animals; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007106:Immune Sera; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D012265:Ribonucleotides / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism
D007106:Immune Sera; D012265:Ribonucleotides; D019204:GTP-Binding Proteins