Abstract
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.
Highlights
Peroxisomes are organelles bounded by a single membrane which are present in almost all eukaryotic cells
Localization of PMP70 in H-4-II-E Cells—In order to investigate the intracellular transport of PMP70 in cultured cells, we first examined whether H-4-II-E cells contain PMP70 in the peroxisomes
A band corresponding to PMP70 was enriched in a light mitochondrial fraction and the distribution of the polypeptide was similar to that of catalase, a peroxisomal marker enzyme (Fig. 1, A and B)
Summary
Peroxisomes are organelles bounded by a single membrane which are present in almost all eukaryotic cells. It has recently been suggested that peroxisomes are formed by post-translational import of newly synthesized proteins into pre-existing peroxisomes, which divide [3,4,5]. We developed an in vitro system based on the import of radiolabeled acyl-CoA oxidase (AOx) into purified rat liver peroxisomes and showed that ATP hydrolysis was required for the translocation of AOx through peroxisomal membranes [9]. Peroxisome assembly factor 1, a peroxisomal integral membrane protein with molecular mass of 35 kDa was cloned and sequenced [23]. Other laboratories, have shown that PMP70, -26, and -22 were synthesized on free polysomes (24 –26), suggesting that the PMPs are likely to be inserted post-translationally into peroxisomal membranes. The occurrence of this event should be confirmed before establishing an in vitro import system
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