Biochemical and Biophysical Research Communications
Studies on the organization of the docking complex involved in matrix protein import into glycosomes of Trypanosoma brucei
Highlights
► Trypanosomatids contain two very different PEX13s in their glycosomal membrane. ► Both PEX13 isoforms are essential for glycosomal matrix-protein import. ► Only trypanosomatids are known to possess a PEX13 isoform with a PTS1. ► The PTS1 of TbPEX13.1 is at the cytosolic face of the glycosomal membrane. ► Both PEX13 mediate PEX14’s correct association with the glycosomal membrane.
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].
Section snippets
Trypanosomes, growth conditions and transfection
Procyclic (i.e. the life-cycle stage living in the tsetse fly’s midgut) forms of T. brucei strain Lister 427, cell line 449 [21], that were used in this study, constitutively express the Escherichia coli tetracycline (Tet) repressor gene from the chromosomally integrated plasmid pHD449, also endowing phleomycin resistance. This cell line is metabolically indistinguishable from the wild type. Procyclic trypanosomes were grown in SDM79 medium [22] supplemented with 15% fetal calf serum and 0.5 μg mL
Results and discussion
TbPEX13.1 is an integral membrane protein [16], and its orthologues similarly predicted so in all trypanosomatids for which genome sequencing has been performed. Nonetheless, it possesses a PTS1 motif, i.e. a consensus targeting motif for a glycosomal matrix protein. Although the tripeptide TKL is rarely used for peroxisome/glycosome targeting, it has been shown to be highly efficient in routing a reporter protein to glycosomes in transient expression experiments of transfected trypanosomes [27]
Acknowledgments
This research was supported through grants to PAMM from the ‘Fonds de la Recherche Scientifique’ (F.R.S-FNRS) of the ‘Communauté française de Belgique’ and the Interuniversity Attraction Poles – Belgian Federal Office for Scientific, Technical and Cultural Affairs. EV and MGL each acknowledge a PhD scholarship from the ‘Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agriculture’ (FRIA) and the Université catholique de Louvain. We thank Dr Frédéric Bringaud (Université
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2022, Frontiers in Cell and Developmental Biology
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Both the authors who contributed equally to this work.