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PEX5 and ubiquitin dynamics on mammalian peroxisome membranes.

ABSTRACT: Peroxisomes are membrane-bound organelles within eukaryotic cells that post-translationally import folded proteins into their matrix. Matrix protein import requires a shuttle receptor protein, usually PEX5, that cycles through docking with the peroxisomal membrane, ubiquitination, and export back into the cytosol followed by deubiquitination. Matrix proteins associate with PEX5 in the cytosol and are translocated into the peroxisome lumen during the PEX5 cycle. This cargo translocation step is not well understood, and its energetics remain controversial. We use stochastic computational models to explore different ways the AAA ATPase driven removal of PEX5 may couple with cargo translocation in peroxisomal importers of mammalian cells. The first model considered is uncoupled, in which translocation is spontaneous, and does not immediately depend on PEX5 removal. The second is directly coupled, in which cargo translocation only occurs when its PEX5 is removed from the peroxisomal membrane. The third, novel, model is cooperatively coupled and requires two PEX5 on a given importomer for cargo translocation--one PEX5 with associated cargo and one with ubiquitin. We measure both the PEX5 and the ubiquitin levels on the peroxisomes as we vary the matrix protein cargo addition rate into the cytosol. We find that both uncoupled and directly coupled translocation behave identically with respect to PEX5 and ubiquitin, and the peroxisomal ubiquitin signal increases as the matrix protein traffic increases. In contrast, cooperatively coupled translocation behaves dramatically differently, with a ubiquitin signal that decreases with increasing matrix protein traffic. Recent work has shown that ubiquitin on mammalian peroxisome membranes can lead to selective degradation by autophagy, or 'pexophagy.' Therefore, the high ubiquitin level for low matrix cargo traffic with cooperatively coupled protein translocation could be used as a disuse signal to mediate pexophagy. This mechanism may be one way that cells could regulate peroxisome numbers.

SUBMITTER: Brown AI

PROVIDER: S-EPMC3894153 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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PEX5 and ubiquitin dynamics on mammalian peroxisome membranes.

Brown Aidan I AI Kim Peter K PK Rutenberg Andrew D AD

PLoS computational biology 20140116 1

Peroxisomes are membrane-bound organelles within eukaryotic cells that post-translationally import folded proteins into their matrix. Matrix protein import requires a shuttle receptor protein, usually PEX5, that cycles through docking with the peroxisomal membrane, ubiquitination, and export back into the cytosol followed by deubiquitination. Matrix proteins associate with PEX5 in the cytosol and are translocated into the peroxisome lumen during the PEX5 cycle. This cargo translocation step is n ...[more]

PMID: 24453954

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Project description:A uniquely sensitive method for ceramide domain detection allowed us to study in detail cholesterol-ceramide interactions in lipid bilayers with low (physiological) ceramide concentrations, ranging from low or no cholesterol (a situation similar to intracellular membranes, such as endoplasmic reticulum) to high cholesterol (similar to mammalian plasma membrane). Diverse fluorescence spectroscopy and microscopy experiments were conducted showing that for low cholesterol amounts ceramide segregates into gel domains that disappear upon increasing cholesterol levels. This was observed in different raft (sphingomyelin/cholesterol-containing) and non-raft (sphingomyelin-absent) membranes, i.e. mimicking different types of cell membranes. Cholesterol-ceramide interactions have been described mainly as raft sphingomyelin-dependent. Here sphingomyelin independence is demonstrated. In addition, ceramide-rich domains re-appear when either cholesterol is converted by cholesterol oxidase to cholestenone or the temperature is decreased. Ceramide is more soluble in cholesterol-rich fluid membranes than in cholesterol-poor ones, thereby increasing the chemical potential of cholesterol. Ceramide solubility depends on the average gel-fluid transition temperature of the remaining membrane lipids. The inability of cholestenone-rich membranes to dissolve ceramide gel domains shows that the cholesterol ordering and packing properties are fundamental to the mixing process. We also show that the solubility of cholesterol in ceramide domains is low. The results are rationalized by a ternary phospholipid/ceramide/cholesterol phase diagram, providing the framework for the better understanding of biochemical phenomena modulated by cholesterol-ceramide interactions such as cholesterol oxidase activity, lipoprotein metabolism, and lipid targeting in cancer therapy. It also suggests that the lipid compositions of different organelles are such that ceramide gel domains are not formed unless a stress or pathological situation occurs.

Dataset Information

PEX5 and ubiquitin dynamics on mammalian peroxisome membranes.

Publications

PEX5 and ubiquitin dynamics on mammalian peroxisome membranes.

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