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.
Highlights
Peroxisomes are single membrane organelles found in most eukaryotic cells [1]
Cargo matrix proteins are shuttled to the peroxisomal membrane, but the only source of energy that has been identified to translocate the cargo into the peroxisome is consumed during the removal of the shuttle protein
How shuttle removal and cargo translocation are coupled energetically has been difficult to determine directly, so we investigate how different models of coupling would affect the measurable levels of ubiquitin on mammalian peroxisomes
Summary
Peroxisomes are single membrane organelles found in most eukaryotic cells [1]. Peroxisomes are highly dynamic organelles, changing their numbers based on the specific metabolic needs of different tissues and cell types [4]. In yeast, changing the carbon source to oleic acid from glucose induces the rapid proliferation of peroxisomes [4]. Changing the carbon source from oleic acid back to glucose results in the decrease of peroxisome numbers in yeast within several hours [4,8]. It has been hypothesized that sufficient ubiquitination of peroxisomal membrane proteins induces pexophagy by recruiting sufficient autophagy receptors such as NBR1 to peroxisomes [12,13]. One candidate is the matrix shuttle protein PEX5, as preventing its recruitment to peroxisomes prevents
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