Peroxisomal Pex11 is a pore-forming protein homologous to TRPM channels.

More than 30 proteins (Pex proteins) are known to participate in the biogenesis of peroxisomes-ubiquitous oxidative organelles involved in lipid and ROS metabolism. The Pex11 family of homologous proteins is responsible for division and proliferation of peroxisomes. We show that yeast Pex11 is a pore-forming protein sharing sequence similarity with TRPM cation-selective channels. The Pex11 channel with a conductance of Λ=4.1 nS in 1.0M KCl is moderately cation-selective (PK(+)/PCl(-)=1.85) and resistant to voltage-dependent closing. The estimated size of the channel's pore (r~0.6 nm) supports the notion that Pex11 conducts solutes with molecular mass below 300-400 Da. We localized the channel's selectivity determining sequence. Overexpression of Pex11 resulted in acceleration of fatty acids ß-oxidation in intact cells but not in the corresponding lysates. The ß-oxidation was affected in cells by expression of the Pex11 protein carrying point mutations in the selectivity determining sequence. These data suggest that the Pex11-dependent transmembrane traffic of metabolites may be a rate-limiting step in the ß-oxidation of fatty acids. This conclusion was corroborated by analysis of the rate of ß-oxidation in yeast strains expressing Pex11 with mutations mimicking constitutively phosphorylated (S165D, S167D) or unphosphorylated (S165A, S167A) protein. The results suggest that phosphorylation of Pex11 is a mechanism that can control the peroxisomal ß-oxidation rate. Our results disclose an unexpected function of Pex11 as a non-selective channel responsible for transfer of metabolites across peroxisomal membrane. The data indicate that peroxins may be involved in peroxisomal metabolic processes in addition to their role in peroxisome biogenesis.

The phosphoinositide 3-kinase Vps34p is required for pexophagy in Saccharomyces cerevisiae.

PIds (phosphoinositides) are phosphorylated derivatives of the membrane phospholipid PtdIns that have emerged as key regulators of many aspects of cellular physiology. We have discovered a PtdIns3P-synthesizing activity in peroxisomes of Saccharomyces cerevisiae and have demonstrated that the lipid kinase Vps34p is already associated with peroxisomes during biogenesis. However, although Vps34 is required, it is not essential for optimal peroxisome biogenesis. The function of Vps34p-containing complex I as well as a subset of PtdIns3P-binding proteins proved to be mandatory for the regulated degradation of peroxisomes. This demonstrates that PtdIns3P-mediated signalling is required for pexophagy.

An involvement of yeast peroxisomal channels in transmembrane transfer of glyoxylate cycle intermediates.

The separate localization of glyoxylate cycle enzymes in the peroxisomes and the cytosol of the yeast Saccharomyces cerevisiae indicates that the peroxisomal membrane must permit the flow of metabolites between the two compartments. The transfer of these metabolites may require peroxisomal membrane channel(s). We used an electrophysiological approach (reconstitution assay in lipid bilayers) to assess the ability of peroxisomal membrane channels to conduct different solutes including metabolites of the glyoxylate cycle. At least two distinct channel-forming activities were detected in peroxisomal preparations. One of these activities was highly inducible by dithiothreitol and showed large-amplitude current increments when 1M KCl was used as a bath solution. Single-channel analysis revealed that the inducible channel is anion-selective (P(Cl(-)) / P(K(+)) = 2.6; P(citrate)/P(K(+)) = 1.6) and displays flickering at holding potentials over + or - 30mV directed upward or downward relative to the main open state of the channel. The channel inducible by DTT facilitates the transfer of solutes with a molecular mass up to 400Da, sufficient to allow the transmembrane trafficking of glyoxylate cycle intermediates between the peroxisomal lumen and the cytoplasm.

Channel-forming activities of peroxisomal membrane proteins from the yeast Saccharomyces cerevisiae.

Highly-purified peroxisomes from the yeast Saccharomyces cerevisiae grown on oleic acid were investigated for the presence of channel (pore)-forming proteins in the membrane of these organelles. Solubilized membrane proteins were reconstituted in planar lipid bilayers and their pore-forming activity was studied by means of multiple-channel monitoring or single-channel analysis. Two abundant pore-forming activities were detected with an average conductance of 0.2 and 0.6 nS in 1.0 m KCl, respectively. The high-conductance pore (0.6 nS in 1.0 m KCl) is slightly selective to cations (P(K+)/P(Cl-) approximately 1.3) and showed an unusual flickering at elevated (> +/-40 mV) holding potentials directed upward relative to the open state of the channel. The data obtained for the properties of the low-conductance pore (0.2 nS in 1.0 m KCl) support the notion that the high-conductance channel represents a cluster of two low-conductance pores. The results lead to conclusion that the yeast peroxisomes contain membrane pore-forming proteins that may aid the transfer of small solutes between the peroxisomal lumen and cytoplasm.