Studies on the organization of the docking complex involved in matrix protein import into glycosomes of Trypanosoma brucei

Abstract

Trypanosoma brucei contains peroxisome-like organelles designated glycosomes because they sequester the major part of the glycolytic pathway. Import of proteins into the peroxisomal matrix involves a protein complex associated with the peroxisomal membrane of which PEX13 is a component. Two very different PEX13 isoforms have recently been identified in T. brucei. A striking feature of one of the isoforms, TbPEX13.1, is the presence of a C-terminal type 1 peroxisomal-targeting signal (PTS1), the tripeptide TKL, conserved in its orthologues in all members of the Trypanosomatidae family so far studied, but absent from TbPEX13.2 and the PEX13s in all other organisms. Despite their differences, both TbPEX13s function as part of a docking complex for cytosolic receptors with bound matrix proteins to be imported. We further characterized TbPEX13.1’s function in glycosomal matrix-protein import. It provides a frame to anchor another docking complex component, PEX14, to the glycosomal membrane or information to correctly position it within the membrane. To investigate the possible function of the C-terminal TKL, we determined the topology of the C-terminal half of TbPEX13.1 in the membrane and show that its SH3 domain, located immediately adjacent to the PTS1, is at the cytosolic face.

Introduction

Protists grouped in the order Kinetoplastea possess organelles called glycosomes because they have the peculiarity to harbor the major part of the glycolytic pathway [1], [2]. Based on morphological data and the fact that additional enzymes found in glycosomes are typical peroxisomal, these organelles are considered as members of the organelle family of peroxisomes. Moreover, for kinetoplastids such as Trypanosoma brucei, the parasite responsible for African sleeping sickness in humans and similar diseases in other mammals, it has been shown that glycosome biogenesis requires the function of proteins homologous to the peroxins (indicated with the acronym PEX) involved in peroxisome biogenesis in other organisms [3], [4]. Trypanosomes, transmitted by tsetse flies, live in the blood of their mammalian hosts. Glycolysis is essential for the parasites when living in the bloodstream because glucose degradation is their sole source of free energy at this stage of their life cycle [5]. The proper compartmentalization of glycolytic enzymes inside glycosomes is also crucial for parasite survival [6], [7], [8].

Like for peroxisomes, glycosomal matrix and membrane proteins are encoded by nuclear genes, synthesized in the cytosol and post-translationally imported into the organelles. Peroxisomal or glycosomal matrix proteins contain a peroxisomal-targeting signal (PTS). Two main targeting signals have been described and are relatively well conserved between species; the type 1 PTS (PTS1) is a C-terminal tripeptide with the general consensus sequence (S/C/A)-(K/R/H)-(L/M) [9]. It is recognized by the cytosolic receptor protein PEX5. The PTS2 is a nonapeptide located near the N-terminus of proteins to be imported and corresponds to the sequence [(R/K)-(L/V/I/Q)-xx-(L/V/I/H)-(L/S/G/A)-x-(H/Q)-(L/A)] [10]. PTS2 motifs are recognized by PEX7, also cytosolically located. After recognition and binding their cargo in the cytosol, the loaded PEX5 and PEX7 steer for the peroxisomes and interact with a docking complex present in the membrane and that in yeasts has been shown to comprise minimally two peroxins, PEX13 and PEX14. Peroxisome targeting of membrane proteins follows a different pathway and requires a routing signal that consists of a stretch of basic amino acids and a transmembrane segment [11], [12].

Despite a low sequence identity compared with mammalian or yeasts homologues, T. brucei PEX5, PEX7 and PEX14 have been identified in databases by sequence homology or by PCR with degenerate primers [13], [14], [15]. However, such analyses were unsuccessful to identify PEX13. Recently, two very different PEX13 isoforms have been identified, TbPEX13.1 and TbPEX13.2, by using different bespoke bioinformatics approaches involving pattern searches plus domain analysis and jackhammer database searches, respectively [16], [17]. The proteins display a very low sequence identity with PEX13s from other organisms, but are, like these other PEX13s, predicted to be integral membrane proteins with two transmembrane segments. Indeed, their localization in the glycosomal membrane has been experimentally confirmed. TbPEX13.1, but not TbPEX13.2, shares with other PEX13s a SH3 domain in its C-terminal half. A most remarkable, unique feature of TbPEX13.1 is the presence of a PTS1 motif, a C-terminal TKL, directly adjacent to the SH3 domain of this membrane protein [16]. Strikingly, this TKL sequence is fully conserved in PEX13.1s of other trypanosomatid genera, strongly suggesting an important function.

In Saccharomyces cerevisiae and fibroblasts, PEX13 has been shown to function not only in receptor docking, but also as a membrane anchor or peroxisomal membrane association factor to PEX14 [18], [19], [20]. Previously, we reported that T. brucei PEX14 is peripherally associated with the cytosolic face of the glycosomal membrane [15]. For TbPEX14 and both TbPEX13 isoforms, the involvement in the glycosomal biogenesis process as a membrane docking factors for the cargo-loaded receptors TbPEX5 or TbPEX7 was confirmed by RNAi: glycosomal PTS-containing matrix proteins became mislocalized to the cytosol when the levels of TbPEX14, TbPEX13.1 or TbPEX13.2 were decreased [15], [16], [17].

In this paper, we present the results of some further analysis of T. brucei PEX13.1. We investigated the topology of this peroxin, important for unraveling the function of the PTS1 sequence and show that PEX13.1 plays a role in correctly positioning PEX14 in the parasite’s glycosomal membrane, as was recently also proved for PEX13.2 [17].