A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack.
We have used an in vitro Golgi protein transport assay dependent on high molecular weight (greater than 100 kD) cytosolic and/or peripheral membrane proteins to study the requirements for transport from the cis- to the medial-compartment. Fractionation of this system indicates that, besides the NEM-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), at least three high molecular weight protein fractions from bovine liver cytosol are required. The activity from one of these fractions was purified using an assay that included the second and third fractions in a crude state. The result is a protein of 115-kD subunit molecular mass, which we term p115. Immunodepletion of the 115-kD protein from a purified preparation with mAbs removes activity. Peptide sequence analysis of tryptic peptides indicates that p115 is a "novel" protein that has not been described previously. Gel filtration and sedimentation analysis indicate that, in its native state, p115 is a nonglobular homo-oligomer. p115 is present on purified Golgi membranes and can be extracted with high salt concentration or alkaline pH, indicating that it is peripherally associated with the membrane. Indirect immunofluorescence indicates that p115 is associated with the Golgi apparatus in situ.
Waters
M G
MG
Program of Cellular Biochemistry and Biophysics, Sloan-Kettering Institute, New York 10021.
Clary
D O
DO
Rothman
J E
JE
D000818:Animals; D000911:Antibodies, Monoclonal; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D003600:Cytosol / Q000737:chemistry; D006056:Golgi Apparatus / Q000737:chemistry* / Q000378:metabolism; D008099:Liver / Q000737:chemistry; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D014759:Viral Envelope Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D014759:Viral Envelope Proteins