Insertion of the 70-kDa peroxisomal membrane protein into peroxisomal membranes in vivo and in vitro.
Biosynthesis and intracellular transport of 70-kda peroxisomal membrane protein (pmp70) has been studied in rat hepatoma, h-4-ii-e cells. Pulse-chase analysis showed that a newly synthesized 35S-PMP70 first appeared in the cytosolic fraction and was then transported into the peroxisomal fraction. The half-life of 35S-PMP70 in the cytosolic fraction was approximately 3 min. Integration of 35S-PMP70 into membranes occurred in the peroxisomal fraction and was completed within 30 min. No proteolytic processing of 35S-PMP70 was observed. An in vitro import system was reconstituted to characterize the insertion mechanism of PMP70 into peroxisomes. Peroxisomes isolated from rat liver were incubated at 26 degrees C with [35S]methionine-labeled in vitro translation products of PMP70 mRNA in the presence of the cytosolic fraction. The peroxisomes were reisolated and insertion of 35S-PMP70 into the membrane was analyzed using a Na2CO3 procedure. The binding and insertion of 35S-PMP70 were dependent on temperature and incubation time and was specific for peroxisomes. Pretreatment of peroxisomes with trypsin and chymotrypsin almost abolished the binding and insertion of 35S-PMP70. The translation products contained several truncated 35S-PMP70s. The NH2 terminally truncated 35S-PMP70s, with a molecular mass greater than 50 kDa, bound to and inserted into peroxisomal membranes, whereas truncated 35S-PMP70s smaller than 45 kDa did not. These results suggest that PMP70 is post-translationally transported to peroxisomes without processing and inserted into peroxisomal membranes by a specific mechanism in which a proteinaceous receptor and a certain internal sequence of PMP70 are involved.
Imanaka
T
T
Department of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan.
Shiina
Y
Y
Takano
T
T
Hashimoto
T
T
Osumi
T
T
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D003001:Cloning, Molecular; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008830:Microbodies / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D051381:Rats; D013462:Sulfur Radioisotopes; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D018528:ATP-Binding Cassette Transporters; C489272:Abcd3 protein, rat; D008565:Membrane Proteins; D013462:Sulfur Radioisotopes; D000255:Adenosine Triphosphate; D008715:Methionine