TbRAB1 and TbRAB2 mediate trafficking through the early secretory pathway of Trypanosoma brucei

Abstract

The African trypanosome possesses a total of 16 small GTPases of the Rab family, which are involved in control of various membrane transport events. Recently the roles of these proteins in the endocytosis and recycling of the major surface antigen of the bloodstream form, the variant surface glycoprotein (VSG), have been described but little has been reported on the roles of Rab proteins in exocytic pathways in trypanosomatids. Whilst phylogenetic analysis based on sequence similarity indicates a comparatively well conserved core set of Rab proteins, the evolutionary distance of the trypanosome lineage from crown eukaryote model systems requires direct experimental evidence to support these sequence data. By database searching we identified two further Rab genes, TbRAB1 and TbRAB2, which are the trypanosome sequence orthologues of mammalian Rab1 and Rab2, important mediators of ER to Golgi and intra-Golgi transport processes. A remarkably high level of sequence conservation is retained between the trypanosome and higher eukaryote orthologues. By immunolocalisation we find that both TbRAB1 and TbRAB2 reside on membranes in intimate association with the Golgi complex. By heterologous expression in mammalian cells we also demonstrate conservation of targeting information in the TbRAB1 and TbRAB2 proteins, whilst TbRAB1, but not TbRAB2, can complement a Ypt1^ts conditional mutant in Saccharomyces cerevisiae. The roles of TbRAB1 and TbRAB2 in exocytosis were examined using RNAi. Suppression of TbRAB1 or TbRAB2 was strongly inhibitory to growth and most importantly both TbRAB1 and TbRAB2 were required for normal progression of VSG through the early secretory pathway. These data indicate conservation of function for these proteins between trypanosomes and crown eukaryotes.

Introduction

The majority of proteins that enter the secretory pathway can be considered to belong to one of three distinct topological classes; soluble, transmembrane domain (TMD) anchored or glycosylphosphatidylinositol (GPI)-anchored. Study of exocytic systems in higher eukaryotes suggests specific transport requirements exist for each class and in particular in the early exocytic system. For example, GPI addition is essential for export of GPI proteins to the Golgi complex; only after the GPI-anchor has been attached are proteins allowed to exit the ER [1]. The specificity of this event has been studied intensively in Saccharomyces cerevisiae, where GPI-anchored proteins are transported from the ER to the Golgi complex via a distinct subset of vesicular carriers [2]; sorting is mediated by the Rab GTPase Ypt1, the tethering factor Uso1 and the Sec34/35 complex [3]. In mammalian cells sorting of GPI-anchored and TMD proteins occurs later in the Golgi complex [4].

Whilst in higher eukaryotes GPI-anchored proteins are a minority of the surface protein complement, in trypanosomatids the GPI-anchor represents a dominant mode of membrane protein attachment. For example, the infective bloodstream form stage (BSF) of Trypanosoma brucei expresses at its surface 5 × 10⁶ copies of the GPI-anchored protein variant surface glycoprotein (VSG) [5]. Because VSG is at a higher concentration in the Golgi complex and the cell surface compared to the ER, T. brucei must have efficient mechanisms for the sorting of GPI-anchored proteins [6].

It is not known if trafficking or sorting mechanisms of GPI proteins are identical throughout the eukaryotic lineage or if the bias in membrane attachment reflects distinct adaptations to protein transport systems. VSG is endocytosed from the cell surface via a clathrin-dependent mechanism that does not involve a concentration step [22]. This is an unusual mode of internalisation as in most organisms incorporation into clathrin-coated pits concentrates the protein via interaction with cytoplasmic receptors whilst many GPI-anchored proteins are internalised by clathrin-independent pathways. Endocytosis and recycling of VSG is dependent on TbRAB5A and TbRAB11 [20], [21], [22], [27] but the roles of Rab proteins in the early secretory pathway has not been evaluated. Addition of the GPI-anchor is clearly important for efficient exocytosis of VSG [23], [24], [25].

Small GTPases of the Rab family play central roles in the regulation of vesicular trafficking and are involved in vesicular targeting, tethering and fusion [7], [8], [9]. The family is extensive with at least 60 members identified in mammalian cells, 11 in S. cerevisiae [10], [11] and 16 in T. brucei. Rab proteins can be subdivided into clades, one of which is involved in early exocytic events, principally exit from the ER and progress through the Golgi complex [12]. In metazoans this group consists of Rab1 (A and B isoforms) and Rab2 (also A and B), suggesting a potentially complex network of pathways at the earliest stages of exocytosis [13], [14], [15]. In yeast a single Rab protein, Ypt1, is involved in trafficking between the ER and Golgi complex [16]. Additional to their roles in trafficking, mammalian Rab1A, Rab1B and Rab2A are also required for maintenance of Golgi structure [17], [18], [19]. A recent in silico analysis of the Rab GTPase complement of T. brucei using the completed trypanosome genome indicates a conserved core of Rab proteins that are likely involved in mediating the major exocytic and endocytic pathways (Ackers et al., in press). To date, only one GTPase, TbRABX1, has been implicated with a putative role in early steps of exocytosis [26]; however, re-examination with the complete T. brucei genome sequence indicates that TbRABX1 (formerly designated TbRAB2 [26], systematically named by Ackers et al., in press) is not a true orthologue of mammalian or yeast Rab proteins involved in ER to Golgi transport. Hence essentially no information on the role of Rabs in ER exit is available and the level of complexity associated with Rab function in early secretory events is currently unknown for trypanosomatids. Here we investigate two new trypanosome Rab proteins that are orthologous to Rab1 and Rab2 from mammals in order to gain insight into early protein export events.