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 β-oxidation in intact cells but not in the corresponding lysates. The β-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 β-oxidation of fatty acids. This conclusion was corroborated by analysis of the rate of β-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 β-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.
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.
Peroxisomes are ubiquitous subcellular organelles involved in diverse metabolic activities, ranging from the oxidation of fatty acids, purines, hydroxyacids, alcohols and polyamines to the synthesis of plasmalogens, ketone bodies and bile acids [1,2]. The protein composition of peroxisomes depends on both the species and environmental conditions. For example, the peroxisomes from fungi and plants, but not from mammals, contain enzymes of the glyoxylate cycle that allow the conversion of acetyl-CoA molecules generated mainly by peroxisomal b-oxidation of fatty acids into succinate, which can be used in a variety of reactions, including the biosynthesis of amino acids or carbohydrates [3].A role for peroxisomal membrane as a permeability barrier to solutes has been a matter of debate for more than 40 years. Only recently was a 'consensus' reached on the idea that this membrane is impermeable to bulky solutes such as ATP and the cofactors, NAD ⁄ H ⁄ , NADP ⁄ H ⁄ , CoA and its acyl derivatives [1,4,5]. By contrast, the permeability of the membrane to small solutes, including inorganic ions and organic metabolites, is still a matter of controversy [1,4,5]. For example, contradictory results were obtained concerning the existence of pH [6,7] or Ca 2+ [8,9] gradients across the peroxisomal membrane. Moreover, the assumption that the presence of such gradients confirms the impermeability of the peroxisomal membrane has recently been challenged [5]. Our previous studies on mammalian peroxisomes showed that the membrane of these particles is permeable to small solutes [10] 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 singlechannel 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) 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.Abbreviation VDAC, voltage-dependent anion channel.
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