Introduction How membrane proteins are sorted between different cellular compartments or between domains of continuous membranes has been actively investigated in representatives from bacteria to mammalian cells. Among the protists, specific ultrastructural features and recent progress in genetic manipulation techniques make the mammalian stage of the parasitic protozoon Trypanosoma brucei an interesting model for the elucidation of both basic and unique aspects of protein sorting. Most studies on the trafficking and sorting in this organism have concentrated on proteins modified by glycosylphosphatidylinositol (GPI) residues, e.g. the highly abundant coat protein known as the variant surface glycoprotein (VSG) either in its native form [see summary in Overath and Engstler (Overath and Engstler, 2004)] or modified versions (Böhme and Cross, 2002; Wang et al., 2003; Triggs and Bangs, 2003) and the transferrin receptor (Mußmann et al., 2004; Mußmann et al., 2003). In contrast, little is known about the sorting of transmembrane proteins. Here we describe a type I membrane protein that can be used for investigating mechanisms of intracellular sorting as well as the molecular traffic between different surface domains. The bloodstream form of T. brucei is an elongated and highly polarized cell, in which the Golgi complex and all endosomal compartments are compactly organized in the posterior region of the cell behind the centrally located nucleus (Fig. 1). Endocytosis and exocytosis are restricted to the flagellar pocket (FP) membrane, which is formed by an invagination of the plasma membrane at the base of the flagellum and constitutes, together with its carbohydrate-rich matrix, a concealed yet extracellular compartment. The flagellar pocket membrane is continuous with both the membrane of the flagellum (F) and the pellicular cell surface (PM). Therefore, the surface is divided in three sub-domains. The mechanisms regulating the composition and dynamics of membrane components between the flagellar pocket membrane 2105 In the parasitic protozoan Trypanosoma brucei, endocytosis and exocytosis occur exclusively at an invagination of the plasma membrane around the base of the flagellum, called the flagellar pocket, which actively communicates by vesicular membrane flow with cisternal/tubulovesicular endosomes. The division of the cell surface into three morphologically distinct sub-domains and the rapid plasma membrane turnover establishes T. brucei as an interesting model for investigations on the sorting and recycling of membrane proteins. In this study we show that the type I membrane protein TbMBAP1, an L-(+)-tartrate- sensitive acid phosphatase, is present in all endosomal membranes but is virtually absent from the lysosome membrane (where this type of protein is mainly found in other organisms) and is not detectable at the cell surface. The endosomal localization of TbMBAP1 is a function of protein abundance. Moderate overexpression (three- to fourfold) leads to an increased appearance within the flagellar pocket membrane. At higher levels the protein is found in the flagellum, and routing to the pellicular plasma membrane is observed at levels 10- to 25-fold above that of wild type. In other organisms L-(+)-tartrate-sensitive acid phosphatases appear to be dispensable but TbMBAP1 is essential, as shown by RNA interference, which causes growth arrest followed by cell death. Comparison of the phenotype of TbMBAP1-depleted cells with that of cells in which endocytosis or exocytosis has been specifically inhibited by RNAi against clathrin of RAB11, reveals that TbMBAP1 is essential for both incoming and recycling membrane traffic. During differentiation of the organism from bloodstream to insect stage, TbMBAP1 is down- regulated and differentially modified in parallel with a 10- fold decrease in the rate of endocytosis. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/118/10/2105/DC1 Key words: GPI, Transmembrane protein, Sorting, Flagellar pocket, RAB11, Clathrin, Quantitative colocalization, Exocytosis Summary The membrane-bound histidine acid phosphataseTbMBAP1 is essential for endocytosis and membrane recycling in Trypanosoma brucei Markus Engstler 1, *, Frank Weise 2,‡ , Karoline Bopp 1,‡ , Christoph G. Grünfelder 2 , Mark Günzel 1 , Niko Heddergott 1 and Peter Overath 2,§ 1 Ludwig-Maximilians-Universität, Department Biologie I, Genetik, Maria-Ward-Strasse 1a, München, 80638, Germany 2 Max-Planck-Institut für Biologie, Abteilung Membranbiochemie, Corrensstrasse 38,Tübingen, 72076, Germany ‡ These authors contributed equally to this work § Present address: Universität Tübingen, Interfakultäres Institut für Zellbiologie, Abteilung Immunologie, Auf der Morgenstelle 15, Tübingen, 72076, Germany *Author for correspondence: (e-mail: engstler@lrz.uni-muenchen.de) Accepted 15 February 2005 Journal of Cell Science 118, 2105-2118 Published by The Company of Biologists 2005 doi:10.1242/jcs.02327 Research Article Journal฀of฀Cell฀Science