Depletion of GIM5 Causes Cellular Fragility, a Decreased Glycosome Number, and Reduced Levels of Ether-linked Phospholipids in Trypanosomes

Abstract

Microbody division in mammalian cells, trypanosomes, and yeast depends on the PEX11 microbody membrane proteins. The function of PEX11 is not understood, and the suggestion that it affects microbody (peroxisome) numbers in mammals and yeast, because it plays a role in beta-oxidation of fatty acids, is controversial. PEX11 and two PEX11-related proteins, GIM5A and GIM5B, are the predominant membrane proteins of the microbodies (glycosomes) of Trypanosoma brucei. The compartmentation of glycosomal enzymes is essential in trypanosomes. Deletion of the GIM5A gene from the form of the parasite that lives in the mammalian blood has no effect on trypanosome growth, but depletion of GIM5B on a gim5a null background causes death. We show here that procyclic trypanosomes, adapted for life in the Tsetse fly vector, survive without GIM5A and with very low levels of GIM5B. The depleted cells have fewer glycosomes than usual and are osmotically fragile, which is a novel observation for a microbody defect. Thus trypanosomes require both GIM5B and PEX11 for the maintenance of normal glycosome numbers. Procyclic cells lacking GIM5A, like mouse cells partially defective in PEX11, have fewer ether-linked phospholipids, even when GIM5B levels are not reduced. Metabolite measurements on GIM5A/B-depleted bloodstream form trypanosomes suggested a change in the flux through the glycolytic pathway. We conclude that PEX11 family proteins play important roles in determining microbody membrane structure, with secondary effects on a subset of microbody metabolic pathways.Microbodies are single-membrane-bound organelles found in very diverse eukaryotes; examples include the peroxisomes of mammals and yeast, the glyoxysomes and peroxisomes of plants, and the glycosomes of kinetoplastid protists. Microbodies manifest considerable diversity with regard to the enzymes and metabolic pathways they contain: examples include catalase, and enzymes of fatty acid beta-oxidation, alcohol oxidation, ether-lipid biosynthesis, and, in the kinetoplastids, glycolysis and glycerol metabolism.

An ancestral role in peroxisome assembly is retained by the divisional peroxin Pex11 in the yeast Yarrowia lipolytica

“…T. brucei GIM5A and GIM5B share 13% and 14% sequence identity with T. brucei Pex11p (Voncken et al, 2003). Knockdown of GIM5 expression resulted in decreased numbers of larger glycosomes, whereas overexpression of GIM5 resulted in increased numbers of smaller glycosomes (Maier et al, 2001;Voncken et al, 2003). However, we did not find strong support for kinetoplastid Pex11p and GIM5 proteins being homologous, as reciprocal pHMMer searches into kinetoplastid genomes retrieved only the query sequence.…”

“…An evolutionary relationship was proposed to exist between GIM5 and trypanosome Pex11p on the basis of sequence similarity and common function (Voncken et al, 2003). T. brucei GIM5A and GIM5B share 13% and 14% sequence identity with T. brucei Pex11p (Voncken et al, 2003). Knockdown of GIM5 expression resulted in decreased numbers of larger glycosomes, whereas overexpression of GIM5 resulted in increased numbers of smaller glycosomes (Maier et al, 2001;Voncken et al, 2003).…”

“…1). An evolutionary relationship was proposed to exist between GIM5 and trypanosome Pex11p on the basis of sequence similarity and common function (Voncken et al, 2003). T. brucei GIM5A and GIM5B share 13% and 14% sequence identity with T. brucei Pex11p (Voncken et al, 2003).…”

Characterization and Developmentally Regulated Localization of the Mitochondrial Carrier Protein Homologue MCP6 from Trypanosoma brucei

“…The restriction enzyme sites SacI, SpeI, BamHI, and ApaI, used for subsequent cloning, are underlined. As described previously for the construction of other T. brucei double-knockout cell lines (33,41,85), using the targeted gene replacement method, these targeting DNA sequences were inserted on either side of NEO and BSD genes bearing actin 5Ј splice sites and actin 3Ј untranslated regions, resulting in a NEO-and BSD-bearing double-knockout (dKO) construct, respectively (see Fig. 5A).…”

PEX11 family members are membrane elongation factors that coordinate peroxisome proliferation and maintenance

“…In addition, the hydrophobic region close to the Ctermini might contribute to trafficking events associated with their correct insertion into the peroxisomal membrane. In lower eukaryotes, physiological levels of PEX11 are sufficient to cause fragmentation of peroxisomes, and peroxisomes are enlarged in cells lacking PEX11 (Erdmann and Blobel, 1995;Voncken et al, 2003). In human cells, high levels of PEX11 proteins lead to tubulation and elongation of the peroxisomal membrane (Figs 3, 4).…”