A C-Terminally Truncated Variant of Neurospora crassa VDAC Assembles Into a Partially Functional Form in the Mitochondrial Outer Membrane and Forms Multimers in vitro.

Fraser G Ferens,Ameet Bharaj,Jörg Stetefeld,Deborah A Court,William A T Summers

doi:10.3389/fphys.2021.739001

Fraser G Ferens, Ameet Bharaj + Show 3 more

Open Access

https://doi.org/10.3389/fphys.2021.739001

Copy DOI

Abstract

The voltage-dependent anion-selective channel (VDAC) is a porin in the mitochondrial outer membrane (MOM). Unlike bacterial porins, several mitochondrial β-barrels comprise an odd number of β-strands, as is the case for the 19-β-stranded VDAC. Previously, a variant of a VDAC from Neurospora crassa, VDAC-ΔC, lacking the predicted 19th β-strand, was found to form gated, anion-selective channels in artificial membranes. In vivo, the two C-terminal β-strands (β18 and β19) in VDAC form a β-hairpin necessary for import from the cytoplasm into mitochondria and the β-signal required for assembly in the mitochondrial outer membrane resides in β19. The current study demonstrated that the putative 18-stranded β-barrel formed by VDAC-ΔC can be imported and assembled in the MOM in vivo and can also partially rescue the phenotype associated with the deletion of VDAC from a strain of N. crassa. Furthermore, when expressed and purified from Escherichia coli, VDAC-ΔC can be folded into a β-strand-rich form in decyl-maltoside. Size exclusion chromatography (SEC) alone or combined with multi-angle light scattering (SEC-MALS) and analytical ultracentrifugation revealed that, unlike full-length VDACs, VDAC-ΔC can self-organize into dimers and higher order oligomers in the absence of sterol.

Highlights

Voltage-dependent anion-selective channels, or mitochondrial porins, reside in the mitochondrial outer membrane and act as general channels that allow the bidirectional flow of metabolites across the membrane [MOM, reviewed in Young et al (2007)]
A comparison of the voltage-dependent anion-selective channel (VDAC) isolated in decyl-maltoside (DM)solubilized mitochondrial membranes of Neurospora crassa and the equivalent protein expressed in E. coli and folded in the same detergent support the equivalence of the native and recombinant forms (Ferens et al, 2019)
An N. crassa strain in which the POR1 gene is replaced by that for VDAC- C was constructed by replacing the wild-type gene in FGSC 9718 (Colot et al, 2006), with the cDNA for the 269-283 Por truncation variant (Popp et al, 1996) downstream of a hygromycin-resistance gene for selection (Colot et al, 2006) (Supplementary Tables 1, 2)

Summary

Introduction

Voltage-dependent anion-selective channels, or mitochondrial porins, reside in the mitochondrial outer membrane and act as general channels that allow the bidirectional flow of metabolites across the membrane [MOM, reviewed in Young et al (2007)]. Voltage-dependent anion-selective channel pores are modulated by interactions with multiple metabolites, including NADH (Zizi et al, 1994) and proteins such as tubulin (Rostovtseva et al, 2008), thereby contributing to the regulation of cellular metabolism (reviewed in Lemasters and Holmuhamedov, 2006; Rostovtseva and Bezrukov, 2008; Caterino et al, 2017; Shoshan-Barmatz et al, 2017; Magri et al, 2018; De Pinto, 2021). Unlike all characterized bacterial β-barrel proteins, which are composed of an even number of β-strands (Wimley, 2003), mammalian (Bayrhuber et al, 2008; Hiller et al, 2008; Ujwal et al, 2008) and zebra fish (Schredelseker et al, 2014) VDAC, expressed in Escherichia coli and folded in detergent, each formed a 19-stranded β-barrel with an N-terminal region that does not contribute to the barrel. A comparison of the VDAC isolated in decyl-maltoside (DM)solubilized mitochondrial membranes of Neurospora crassa and the equivalent protein expressed in E. coli and folded in the same detergent support the equivalence of the native and recombinant forms (Ferens et al, 2019)

Methods

Results

Conclusion