INSERTION OF THE 70-KDA PEROXISOMAL MEMBRANE-PROTEIN INTO PEROXISOMALMEMBRANES IN-VIVO AND IN-VITRO

Biosynthesis and intracellular transport of 70-kDa peroxisomal membran e protein (PMP70) has been studied in rat hepatoma, H-4-II-E cells. Pu lse-chase analysis showed that a newly synthesized S-35-PMP70 first ap peared in the cytosolic fraction and was then transported into the per oxisomal fraction. The half-life of S-35-PMP70 in the cytosolic fracti on was approximately 3 min. Integration of S-35-PMP70 into membranes o ccurred in the peroxisomal fraction and was completed within 30 min. N o proteolytic processing of S-35-PMP70 was observed. An in vitro impor t system was reconstituted to characterize the insertion mechanism of PMP70 into peroxisomes. Peroxisomes isolated from rat liver were incub ated at 26 degrees C with [S-35]methionine-labeled in vitro translatio n products of PMP70 mRNA in the presence of the cytosolic fraction. Th e peroxisomes were reisolated and insertion of S-35-PMP70 into the mem brane was analyzed using a Na2CO3 procedure. The binding and insertion of S-35-PMP70 were dependent on temperature and incubation time and w as specific for peroxisomes, Pretreatment of peroxisomes with trypsin and chymotrypsin almost abolished the binding and insertion of S-35-PM P70, The translation products contained several truncated S-35-PMP70s. The NH2 terminally truncated S-35-PMP70s, with a molecular mass great er than 50 kDa, bound to and inserted into peroxisomal membranes, wher eas truncated S-35-PMP70s smaller than 45 kDa did not, These results s uggest that PMP70 is post-translationally transported to peroxisomes w ithout processing and inserted into peroxisomal membranes by a specifi c mechanism in which a proteinaceous receptor and a certain internal s equence of PMP70 are involved.