Characterization of glycosomal RING finger proteins of trypanosomatids

Abstract

The glycosomes of trypanosomatids are essential organelles that are evolutionarily related to peroxisomes of other eukaryotes. The peroxisomal RING proteins -PEX2, PEX10 and PEX12 -comprise a network of integral membrane proteins that function in the matrix protein import cycle. Here, we describe PEX10 and PEX12 in Trypanosoma brucei, Leishmania major, and Trypanosoma cruzi. We expressed GFP fusions of each T. brucei coding region in procyclic form T. brucei, where they localized to glycosomes and behaved as integral membrane proteins. Despite the weak transmembrane predictions for TbPEX12, protease protection assays demonstrated that both the N and C termini are cytosolic, similar to mammalian PEX12. GFP fusions of T. cruzi PEX10 and L. major PEX12 also localized to glycosomes in T. brucei indicating that glycosomal membrane protein targeting is conserved across trypanosomatids.

Conservation of PEX19-Binding Motifs Required for Protein Targeting to Mammalian Peroxisomal and Trypanosome Glycosomal Membranes

“…The cytosolic phosphoglycerate kinase was found in the digitonin supernatant, whereas the glycosomal form partitioned to the digitonin pellet. From there, it associated predominantly to the carbonate supernatant fraction, although some fractionated with the carbonate pellet as observed previously (45). As shown in Fig.…”

“…4A. However, another PMP, the RING finger protein TbPEX12 (29,45), does not seem to follow this prediction pattern, as it did not show binding of its highest scoring regions to GST-TbPEX19. We therefore assessed whether this protein would target to human peroxisomes.…”

“…Following centrifugation, the organellar pellet was treated with 0.2 M sodium carbonate, pH 11, and the integral and peripheral membrane protein fractions were separated by centrifugation as described previously (45). Cell equivalents of the digitonin supernatant, carbonate supernatant (matrix and peripheral membrane proteins), and pellets (integral membrane proteins) were subjected to immunoblot analysis.…”

“…For a list of all primers used in plasmid construction, see Table S1 in the supplemental material. The TbPEX19 RNAi plasmid was described previously (5), as were the T. brucei expression plasmids encoding N-terminal GFP fusions to TbPEX10 and TbPEX12 (45). For the expression of N-terminal GFP fusion proteins in human fibroblasts, these genes were amplified from their respective plasmids, cloned into pGEM T-Easy, sequenced, and cloned into the pEGFP-C1 plasmid.…”

“…Here, we examine PMP targeting by PEX19 via comparative analysis of this process in T. brucei and human cells. We have previously shown that cross-species targeting of PEX10 and PEX12 is conserved among trypanosomatids (45), and therefore we focus the work here on T. brucei as the representative organism for this group. We demonstrate here that although many aspects of PMP trafficking have remained conserved over the course of evolution, subtle differences that may prove useful in fighting trypanosomatid diseases exist.…”