Translocation of acyl-CoA oxidase into peroxisomes requires ATP hydrolysis but not a membrane potential.

Abstract

Abstract. An efficient system for the import of newly synthesized proteins into highly purified rat liver peroxisomes was reconstituted in vitro. 35S-Labeled acyl-CoA oxidase (AOx) was incorporated into peroxisomes in a proteinase K-resistant fashion. This import was specific (did not occur with mitochondria) and was dependent on temperature, time, and peroxisome concentration. Under optimal conditions •30% of T hE biogenesis ofperoxisomes has unique features that distinguish it from the assembly of other organelles (21). In rat liver, for example, the peroxisomal matrix proteins, the core protein urate oxidase, and a major 22-kD integral membrane protein are all synthesized on free polyribosomes. Newly synthesized polypeptides are initially located in the cell cytosol and subsequently appear in peroxisomes, as shown by in vivo pulse-chase experiments. These findings imply the posttranslational import of newly synthesized polypeptides by preexisting peroxisomes. In this respect, peroxisome assembly resembles that of mitochondria and chloroplasts, although peroxisomes have only one membrane. In contrast to most other organelle proteins, peroxisomal proteins are generally synthesized at their final sizes and are not processed proteolytically upon import. Moreover, topogenic information is present in the carboxy-terminal region of the one peroxisomal protein so far studied (38).Practically no information is yet available on the details of the import mechanism, including the energy requirements (21). Bellion and Goodman have recently reported (2) that carbonylcyanide-m-chlorophenylhydrazone (CCCP) ~ prevents the import of alcohol oxidase into Candida boidinii peroxisomes. This effect is puzzling in view of the fact that peroxisomal membranes contain pores that allow the passage of molecules as large as 800 D (40). A requirement for ATP has emerged recently as a common factor in posttranslational Portions of this work have appeared in abstract form (1986. Eur. J. CellBiol.

Quantitative Analysis of Peroxisomal Protein Import in Vitro

“…All assays are in accord that peroxisomes do not import substrates at low temperatures ( [16,18,20], Figs. 3 and 4B).…”

“…Import appeared to peak at approximately 45 min-roughly consistent with the time course results obtained with other import assays. That is, Wendland and Subramani [20], using the immunofluorescence-based import assay, and Walton et al [4], using the microinjection-based import assay, observed maximal import at 1 h; Imanaka et al [16], using purified rat liver peroxisomes, observed a plateau at approximately 30 min.…”

“…In vitro assays employing purified peroxisomes suggest no effect of cytosol [16], whereas the immunofluorescence-based, semipermeabilized system has been shown to require cytosol [20] as well as to be cytosol-independent [28]. Since a number of peroxins and chaperone molecules are cytosolic [14,29], it is reasonable that cytosolic factors are necessary.…”

“…Using protease protection as the hallmark of import, radiolabeled substrates have been incubated in in vitro import reactions with purified rat liver [16] or yeast peroxisomes [17]. In rat liver, these experiments showed that import of the PTS1-containing substrate acyl-CoA oxidase was ATP-, time-, temperature-, and signal-dependent [16].…”

“…In rat liver, these experiments showed that import of the PTS1-containing substrate acyl-CoA oxidase was ATP-, time-, temperature-, and signal-dependent [16]. Unfortunately, organelle instability and questions regarding the import proficiencies of peroxisome subpopulations have prevented the assay from becoming more universally utilized.…”