Organelle-specific phosphorylation. Identification of unique membrane phosphoproteins of the endoplasmic reticulum and endosomal apparatus.
Highly purified endoplasmic reticulum fractions from rat liver and dog pancreas harbor membrane-associated kinases that phosphorylate integral membrane proteins of 90, 56, 35, and 15 kDa with [gamma-32P]GTP and of 90, 56, and 35 kDa with [gamma-32P]ATP. Of these, only the 35-kDa phosphoprotein was N-glycosylated. Screening of Golgi fractions, endosomes, plasma membranes, lysosomes, and mitochondria revealed phosphoproteins unique to each organelle. In particular, endosomes were found to harbor a 48-kDa extrinsic membrane protein and two or more integral membrane phosphoproteins of 30-35 kDa. None of these were N-glycosylated as judged by their insensitivity to digestion by N-glycosidase F and a lack of binding to concanavalin A or wheat germ agglutinin. Since the 30-35 kDa membrane phosphoproteins present in Golgi-free endosomal fractions were not detected in endosome-free, highly purified Golgi fractions and were found exclusively in horseradish peroxidase-containing endosomes as determined by the diaminobenzidine shift protocol, then these membrane phosphoproteins are unique to endosomes. Since membrane phosphoproteins unique to the endoplasmic reticulum have been shown to have important functional significance in calcium binding and as a membrane chaperone(s) (Wada, I., Rindress, D., Cameron, P.H., Ou, W.-J., Doherty, J.-J., II, Louvard, D., Bell, A.W., Dignard, D., Thomas, D.Y., and Bergeron, J.J.M. (1991) J. Biol. Chem. 266, 19599-19610; Ahluwalia, N., Bergeron, J.J.M., Wada, I., Degen, E., and Williams, D.B. (1992) J. Biol. Chem. 267, 10914-10918), then the unique endosomal phosphoproteins may serve equally important functions in addition to serving as novel markers for the organelle.
Rindress
D
D
Department of Anatomy, McGill University, Montreal, Quebec, Canada.
Lei
X
X
Ahluwalia
J P
JP
Cameron
P H
PH
Fazel
A
A
Posner
B I
BI
Bergeron
J J
JJ
D000818:Animals; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D015388:Organelles / Q000378:metabolism*; D010179:Pancreas / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D008565:Membrane Proteins; D010750:Phosphoproteins; D011494:Protein Kinases