Recombinant yeast VDAC2: a comparison of electrophysiological features with the native form

Abstract

Voltage‐dependent anion channel isoform 2 of the yeast Saccharomyces cerevisiae ( yVDAC 2) was believed for many years to be devoid of channel activity. Recently, we isolated yVDAC 2 and showed that it exhibits channel‐forming activity in the planar lipid bilayer system when in its so‐called native form. Here, we describe an alternative strategy for yVDAC 2 isolation, through heterologous expression in bacteria and refolding in vitro . Recombinant yVDAC 2, like its native form, is able to form voltage‐dependent channels. However, some differences between native and recombinant yVDAC 2 emerged in terms of voltage dependence and ion selectivity, suggesting that, in this specific case, the recombinant protein might be depleted of post‐translational modification(s) that occur in eukaryotic cells.

Voltage-Dependent Anion Selective Channel Isoforms in Yeast: Expression, Structure, and Functions

“…The electrophysiology at the PLB revealed that both native and recombinant proteins were able to form pores with the same, typical VDAC-like conductance of ∼3.6 nS in 1 M KCl, as displayed in Figure 2A) (Guardiani et al, 2018;Magrì et al, 2019). As for voltage-dependence, native yVDAC2 resembled yeast or human VDAC1, even if it began to close at ±40-50 mV.…”

“…It was called native yVDAC2. The second strategy applied the heterologous expression of a 6xHis-tagged yVDAC2 in E. coli ( Magrì et al, 2019 ). Being a membrane protein, yVDAC2 localized in the inclusion bodies and needed high concentrations of denaturant to be purified, followed by a refolding step in presence of specific detergents ( Engelhardt et al, 2007 ).…”

“…This suggests that yVDAC2 is slightly less sensitive to the applied voltage ( Figure 2B ). However, this specific aspect was amplified in the recombinant protein, which began to close only at ± 80–90 mV ( Magrì et al, 2019 ). The difference between the native and recombinant forms of yVDAC2 raises the interesting question of whether the native yVDAC2 was subject to specific PTMs, not occurring in the heterologous expression in E. coli .…”

“…Thus, anion selectivity was expected for yVDAC2 in the open state, as also predicted by bioinformatic analysis, with a chloride selectivity estimated 2–3 times higher than that displayed by isoform 1 ( Guardiani et al, 2018 ). The analysis of native yVDAC2 at the PLB allowed the identification of up three states with different parameters of ionic selectivity: two of them appeared to be high-conductance states but with opposite selectivity ( Guardiani et al, 2018 ; Magrì et al, 2019 ). In the open state, the ratio Cl – : K + for yVDAC2 was 3:1, definitely more anionic than the corresponding state displayed by yVDAC1.…”

Renaissance of VDAC: New Insights on a Protein Family at the Interface between Mitochondria and Cytosol

“…Our group expressed the yeast VDAC2 isoform and characterized its activity after reconstitution in planar membranes. We found that recombinant yVDAC2 is also able to fold forming a pore and that its electrophysiological characteristics are very similar to those of yVDAC1 [ 83 , 84 ]. While the expression pattern and the physiological role of yVDAC2 remain unknown, knockout out of yVDAC1 forces cells to adopt a fermentative energy metabolism, highlighting the failure of yVDAC2 to compensate for the lack of yVDAC1 [ 82 ].…”