Integration of porin synthesized in vitro into outer mitochondrial membranes

Abstract

Porin, an intrinsic protein of outer mitochondrial membranes of rat liver, was synthesized in vitro in a cellfree translation system with rat liver RNA. The apparent molecular mass of porin synthesized in vitro was the same as that of its mature form (34 kDa). This porin was post-translationally integrated into the outer membrane of rat liver mitochondria when the cell-free translation products were incubated with mitochonadria at 30 "C even in the presence of a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Therefore, the integration of porin seemed to proceed energy-independently as reported by Eur. J . Biochern. 126, 197-2021. Its integration seemed, however, to require the participation of the inner membrane, since porin was not integrated when isolated outer mitochondrial membranes alone were incubated with the translation products. Porin in the cell-free translation products bound to the outside of the outer mitochondrial membrane when incubated with intact mitochondria at 0°C for 5 min. When the incubation period at 0°C was prolonged to 60 min, this porin was found in the inner membrane fraction, which contained monoamine oxidase, suggesting that porin might bind to a specific site on the outer membrane in contact or fused with the inner membrane (a so-called OM-IM site). This porin bound to the OM-IM site was integrated into the outer membrane when the membrane fraction was incubated at 30°C for 60 min. These observations suggest that porin bound to the outside of the outer mitochondrial membrane is integrated into the outer membrane at the OM-IM site by some temperature-dependent process(es).Porin is a major integral protein of the outer mitochondrial membrane, where it forms a channel for the non-specific passage of low-molecular-mass hydrophilic solutes [l]. Recently, this protein was purified from rat liver [2] and Neurospora crassa [3, 41 and its in vitro synthesis by this organism was reported [5 -71. Since porin can easily be synthesized in vitro in a cell-free translation system with RNA prepared from eukaryotic cells, it seems to be the most useful protein for use in studies on post-translational integration of proteins synthesized in cytoplasmic ribosomes into the outer mitochondrial membrane.In this paper, we report studies on the integration of porin, synthesized in a cell-free translation system, into the outer membrane of mitochondria from rat liver. The sequence of events involved in the integration of porin into the outer mitochondrial membrane is also discussed. MATERIALS AND METHODS AnimalsMale Wistar rats weighing about 200 g were used. Preparation of H-labeled porin and antibody against porinPorin was purified by the method of Freitag et al. 2 mg/ml) at 30°C. The mixture was treated with trypsin (final concentration, 250 Fg/ml) for 20 min at O"C, and the mitochondria were reisolated by centrifugation at 10000 x g for 5 min. The 35S-labeled porin was isolated by immunoprecipitation from the mitochondrial and post-mitochondria1 fraction and analyzed as described previously [ ...

Biogenesis of mitochondrial porin: The import pathway

“…It has been speculated that the membrane potential (negative inside) exerts an electrophoretic effect on the positively charged amino acids contained in the amino-terminal presequences and helps to mediate the translocation across the inner membrane 49. With porin, a membrane potential is not required for the import is, 18,38,43. This is in agreement with the absence of a positive net charge in the amino-terminal portion of the precursor polypeptide which is proposed to be the targeting sequence 35, 39.…”

Purification and Characterization of the Voltage-Dependent Anion-Selective Channel Protein from Wheat Mitochondrial Membranes

“…The electrophysiological properties of these VDACs were found to be almost identical with those of fungal organisms (Colombini, 1979;Colombini, 1983). The respective mammalian protein was purified (Colombini, 1983;De Pinto et al, 1989), and an import experiment of an in vitro translated precursor into mitochondria was performed (Ono and Tuboi, 1987). Moreover, a set of studies was carried out involving VDAC's compartmentation among mitochondria of specific organs and between morphologically different types of mitochondria within the same organ.…”

The N-terminal Domain of Rat Liver Carnitine Palmitoyltransferase 1 Mediates Import into the Outer Mitochondrial Membrane and Is Essential for Activity and Malonyl-CoA Sensitivity

“…The first criterion used was the tight membrane association of the native L-CPT1 (31). Membrane insertion of proteins can be assessed by measuring their levels of resistance to extraction with alkaline buffer (12,32,33). This procedure distinguishes both soluble and peripherally membrane-bound proteins from integral proteins such as porin (33) or Tom70p (12).…”