No abstract

Mitochondrial porins are predicted to traverse the outer membrane as a series of beta-strands, but the precise structure of the resulting beta-barrel has remained elusive. Toward determining the positions of the membrane-spanning segments, a series of small deletions was introduced into several of the predicted beta-strands of the Neurospora crassa porin. Overall, three classes of porin variants were identified: i), those producing large, stable pores, indicating deletions likely outside of beta-strands; ii), those with minimal pore-forming ability, indicating disruptions in key beta-strands or beta-turns; and iii), those that formed small unstable pores with a variety of gating and ion-selectivity properties. The latter class presumably results from a subset of proteins that adopt an alternative barrel structure upon the loss of stabilizing residues. Some variants were not sufficiently stable in detergent for structural analysis; circular dichroism spectropolarimetry of those that were did not reveal significant differences in the overall structural composition among the detergent-solubilized porin variants and the wild-type protein. Several of the variants displayed altered tryptophan fluorescence profiles, indicative of differing microenvironments surrounding these residues. Based on these results, modifications to the existing models for porin structure are proposed.

Mitochondrial porin forms an aqueous pore in the outer membrane, through which selective passage of small metabolites and ions occurs, thereby regulating both mitochondrial function and cellular respiration. Investigations of the structure and function of porin have been performed with whole mitochondria, membrane vesicles, artificial membranes, and in detergent solutions, resulting in numerous models of porin structure. The mechanisms by which this protein functions are undoubtedly linked to its structure, which remained elusive until 2008, with reports of 3 high-resolution structures of this voltage-dependent, anion-selective channel (VDAC). The barrel structure is relatively simple yet unique: it is arranged as 19 anti-parallel beta-strands, with beta-strands 1 and 19 aligned parallel to each other to close the barrel. The N-terminal helical component is located within the lumen of the channel, although its precise structure and location in the lumen varies. With the basic barrel structure in hand, the data obtained in attempts to model the structure and understand porin over the past 20 years can be re-evaluated. Herein, using the mammalian VDAC structures as templates, the amassed electrophysiological and biochemical information has been reassessed with respect to the functional mechanisms of VDAC activity, with a focus on voltage-dependent gating.

Mitochondrial porin, the voltage-dependent anion channel, plays an important role in metabolism and other cellular functions within eukaryotic cells. To further the understanding of porin structure and function, Neurospora crassa wild-type porin was replaced with a deletion variant lacking residues 238-242 (238porin). 238porin was assembled in the mitochondrial outer membrane, but the steady state levels were only about 3% of those of the wild-type protein. The strain harbouring 238porin displayed cytochrome deficiencies and expressed alternative oxidase. Nonetheless, it exhibited an almost normal linear growth rate. Analysis of mitochondrial proteomes from a wild-type strain FGSC9718, a strain lacking porin (ΔPor-1), and one expressing only 238porin, revealed that the major differences between the variant strains were in the levels of subunits of the NADH:ubiquinone oxidoreductase (complex I) of the electron transport chain, which were reduced only in the ΔPor-1 strain. These, and other proteins related to electron flow and mitochondrial biogenesis, are differentially affected by relative porin levels.

Mitochondrial porin, which forms voltage-dependent anion-selective channels (VDAC) in the outer membrane, can be folded into a 19-β-stranded barrel. The N terminus of the protein is external to the barrel and contains α-helical structure. Targeted modifications of the N-terminal region have been assessed in artificial membranes, leading to different models for gating in vitro. However, the in vivo requirements for gating and the N-terminal segment of porin are less well-understood. Using Neurospora crassa porin as a model, the effects of a partial deletion of the N-terminal segment were investigated. The protein, ΔN2-12porin, is assembled into the outer membrane, albeit at lower levels than the wild-type protein. The resulting strain displays electron transport chain deficiencies, concomitant expression of alternative oxidase, and decreased growth rates. Nonetheless, its mitochondrial genome does not contain any significant mutations. Most of the genes that are expressed in high levels in porin-less N. crassa are expressed at levels similar to that of wild type or are slightly increased in ΔN2-12porin strains. Thus, although the N-terminal segment of VDAC is required for complete function in vivo, low levels of a protein lacking part of the N terminus are able to rescue some of the defects associated with the absence of porin.