27] Isolation and properties of the porin of the outer mitochondrial membrane from Neurospora crassa

Abstract

Publisher Summary The pore-forming protein, called mitochondrial porin, is a major component of outer mitochondrial membranes. This chapter describes the isolation and properties of the porin of the outer mitochondrial membrane from Neurospora crassa. Mitochondrial outer membranes are isolated by swelling-shrinking of the mitochondria and subsequent separation by a sucrose step centrifugation. Differential detergent extraction of the outer membrane followed by diethylaminoethyl (DEAE)-cellulose chromatography gives pure porin, which forms active pores after reconstitution into artificial bilayers. Porin isolation from whole mitochondria involves lysis of whole mitochondria with Genapol X-100 followed by chromatography on hydroxyapatite, Celite, and DEAE-cellulose. This procedure gives pure and functionally active porin in high yield and can be easily scaled up. Mitochondrial porin is synthesized on cytoplasmic free polysomes without an additional presequence. In contrast to the biogenesis of mitochondrial inner membrane or matrix proteins, assembly of the porin is not dependent on energization of the mitochondria.