Endocytosis of a Glycosylphosphatidylinositol-anchored Protein via Clathrin-coated Vesicles, Sorting by Default in Endosomes, and Exocytosis via RAB11-positive Carriers

Grünfelder, Christoph G.; Engstler, Markus; Weise, Frank; Schwarz, Heinz; Stierhof, York‐Dieter; Morgan, Gareth; Field, Mark C.; Overath, Peter

doi:10.1091/mbc.e02-10-0640

Cited by 123 publications

(197 citation statements)

References 51 publications

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“…No ultrastructural evidence for dynamin collars on CCVs or pits has been obtained (55), and the finding that ConA endocytosis is unaltered by TbDLP suppression is consistent with this hypothesis. Hence, it appears that the original role of dynamin family proteins was in mitochondrial and chloroplast membrane scission, and that the endocytic and exocytic functions for dynamin have arisen later in evolution.…”

Section: Discussionmentioning

confidence: 75%

The Single Dynamin-like Protein of Trypanosoma brucei Regulates Mitochondrial Division and Is Not Required for Endocytosis

Morgan

Goulding

Field

2004

Journal of Biological Chemistry

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Members of the evolutionarily conserved dynamin-related GTPase family mediate numerous cellular membrane remodeling events. Dynamin family functions include the scission of clathrin-coated pits from the plasma membrane, mitochondrial fission, and chloroplast division. Here we report that the divergent eukaryote Trypanosoma brucei possesses a single dynamin family gene, which we have designated TbDLP. Furthermore, a single dynamin family gene is also found in the Leishmania major and Trypanosoma vivax genomes, indicating that this is a conserved feature among the kinetoplastida. TbDLP is most homologous to the DMN/DRP family of dynamin-like proteins. Indirect immunofluorescence microscopy reveals that TbDLP is distributed in punctate structures within the cell that partially co-localize with the mitochondrion when labeled with MitoTracker. To define TbDLP function, we have used RNA interference to silence the TbDLP gene. Reduction of TbDLP protein levels causes a profound alteration in mitochondrial morphology without affecting the structure of other membrane-bound compartments, including the endocytic and exocytic apparatus. The mitochondrial profiles present in wild type trypanosomes fuse and collapse in the mutant cells, and by electron microscopy the mitochondria are found to contain an accumulation of constriction sites. These findings demonstrate TbDLP functions in division of the mitochondrial membrane. Most significantly, as TbDLP is the sole member of the dynamin family in this organism, scission of clathrin-coated pits involved in protein trafficking through the highly active endocytic system in trypanosomes must function in the absence of dynamin. The evolutionary implications of these findings are discussed.

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Section: Discussionmentioning

confidence: 75%

The Single Dynamin-like Protein of Trypanosoma brucei Regulates Mitochondrial Division and Is Not Required for Endocytosis

Morgan

Goulding

Field

2004

Journal of Biological Chemistry

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“…on VSG trafficking, which have shown that the total VSG complement on the surface of T. brucei is internalized at enormous rate, resulting in a turnover time of 12 min (12,35,43). The fact that it takes more than an hour for the Tf-R to disappear from the surface after the addition of bovine Tf (Fig.…”

Section: Fig 7 Variation Of Tf-r Levels In T Brucei Growing In Culmentioning

confidence: 99%

“…This is unexpected, as trypanosomes are normally able to handle the routing of new VSG efficiently. The Tf-R resembles a VSG dimer in structure and in its glycosylphosphatidylinositol anchor (23), but T. brucei makes at least 100-fold more VSG than Tf-R, and only 11% of this VSG is present intracellularly (12,35,43).…”

Section: Fig 7 Variation Of Tf-r Levels In T Brucei Growing In Culmentioning

confidence: 99%

Factors Affecting the Level and Localization of the Transferrin Receptor in Trypanosoma brucei

Mußmann

Engstler

Gerrits

et al. 2004

Journal of Biological Chemistry

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Transfer of bloodstream-form Trypanosoma brucei variant 221a from calf serum to dog serum-based medium induces acute iron starvation, as the transferrin receptor (Tf-R) of variant 221a binds dog Tf poorly. We show here that transfer to dog serum induces a 3-5-fold increase in Tf-R mRNA and protein within one doubling time (8 h). Because iron stores are still high 8 h after transfer, we infer that the signal for Tf-R overproduction is the decreased availability of cytosolic iron when cellular iron import drops. Up to 30% of the extra Tf-R spills out of the flagellar pocket onto the pellicular surface. Because the 5-fold increase in Tf-R is accompanied by a 5-fold increase in bovine Tf uptake, the up-regulation of Tf-R levels in response to Tf starvation helps the trypanosome to compete for limiting amounts of Tf. We noted that Tf-R levels also vary in calf serum medium. Cells in dense cultures contain up to 5-fold more Tf-R mRNA and protein than in dilute cultures. Only onetenth of the extra Tf-R reaches the pellicular surface. The increase cannot be explained by a lack of Tf or to cell density sensing but is due to pericellular hypoxia. Our results show that bloodstream-form trypanosomes can regulate the expression of the two Tf-R subunit genes and the localization of their gene products in a flexible manner. This flexibility is made possible by the promoter-proximal position of the two genes in the variant surface glycoprotein expression site.The unicellular protozoan parasite Trypanosoma brucei can infect a broad range of mammals. It multiplies extracellularly in blood and escapes elimination by the immune system through antigenic variation of its surface coat, which consists of one major protein species, the variant surface glycoprotein (VSG), 1 attached to the plasma membrane by a glycosylphosphatidylinositol anchor (1, 2). African trypanosomes cover their iron need by taking up host transferrin (Tf) (3-5). Uptake occurs in the flagellar pocket (3, 6, 7), an invagination of the plasma membrane where all endocytosis and traffic to the trypanosome surface takes place (8 -12), and is mediated by a transferrin receptor (Tf-R), a heterodimer consisting of subunits encoded by expression site-associated gene (ESAG) 6 and 7 (13-17), which are located in the telomeric VSG gene expression sites (for review, see .The ESAG6 subunit of about 400 amino acids is attached to the plasma membrane by a glycosylphosphatidylinositol anchor; ESAG7 of about 340 amino acids remains membraneattached by holding on to ESAG6 (for review, see Ref. 22). The trypanosomal Tf-R resembles VSG dimers in apparent structure and (distantly) in sequence (23), and it is unlike other Tf-Rs in nature.

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“…This region is characterized by a narrowing of the FP at its apical end until it is little larger in diameter than the flagellum. Again, the cytoskeleton appears central to this: in particular, an electron-dense structure called the ''collar'' can be seen on the cytoplasmic side of this region, and ablation of a protein that localizes to this structure disrupts FP morphogenesis (7).Because of its central importance to pathogenicity, the endocytic activity of T. brucei has long been studied by using well-defined markers for endocytosis and subcellular compartments (1,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). These studies have established the speed, timing, and routes of uptake and traffic within the cell (1,16,18,20), but a number of critical questions remain unanswered.…”

mentioning

confidence: 99%

“…These studies have established the speed, timing, and routes of uptake and traffic within the cell (1,16,18,20), but a number of critical questions remain unanswered. First, given the constriction of the FP at its junction with the cell body membrane, how do molecules in the extracellular milieu gain access to its lumen?…”

mentioning

confidence: 99%

Membrane domains and flagellar pocket boundaries are influenced by the cytoskeleton in African trypanosomes

Gadelha

Rothery

Morphew

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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A key feature of immune evasion for African trypanosomes is the functional specialization of their surface membrane in an invagination known as the flagellar pocket (FP), the cell's sole site of endocytosis and exocytosis. The FP membrane is biochemically distinct yet continuous with those of the cell body and the flagellum. The structural features maintaining this individuality are not known, and we lack a clear understanding of how extracellular components gain access to the FP. Here, we have defined domains and boundaries on these surface membranes and identified their association with internal cytoskeletal features. The FP membrane appears largely homogeneous and uniformly involved in endocytosis. However, when endocytosis is blocked, receptor-mediated and fluid-phase endocytic markers accumulate specifically on membrane associated with four specialized microtubules in the FP region. These microtubules traverse a distinct boundary and associate with a channel that connects the FP lumen to the extracellular space, suggesting that the channel is the major transport route into the FP.electron tomography ͉ endocytosis ͉ freeze fracture ͉ Trypanosoma brucei ͉ flagellum P arasitic protozoa, like many cells, organize their surface membranes into subdomains or microenvironments that are specialized to accomplish particular recognition, secretory, and endocytic functions. The surfaces of parasitic organisms, however, have an extra level of constraint; they must perform these vital roles while the organism avoids elimination by defensive responses mounted by the host.The compartmentalization of surface membrane is especially interesting in African trypanosomes because of their particular parasitic lifestyle. Trypanosoma brucei is a flagellate protozoan that lives in the blood of mammals in an exclusively extracellular form, fully exposed to the host immune system. Survival is assisted both by rapid endocytosis that clears the entire cell surface of bound antibodies within Ϸ7 min (1) and through the expression of a series of immunologically distinct cell surface coats. Each coat is produced from a single GPI-anchored variant surface glycoprotein (VSG; ref.2). Periodic switching of the expressed VSG gene from a vast silent library enables the parasite to avoid clearance by the host's adaptive immune response, hence prolonging infection and increasing the chances of transmission by the bite of a tsetse fly.African trypanosomes maintain the VSG coat free of most invariant endocytosis receptors that could otherwise elicit an immune response from which the organism could not escape. This is achieved through specialization of the plasma membrane at the base of the flagellum to create a protected invagination, the flagellar pocket (FP), in which the invariant receptors for endocytosis and innate immune evasion are concentrated (3, 4). The FP is the sole site for all endocytosis and exocytosis, and along with this critical function, it also plays important roles in protein and lipid sorting and recycling (for review, see ref. ...

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Endocytosis of a Glycosylphosphatidylinositol-anchored Protein via Clathrin-coated Vesicles, Sorting by Default in Endosomes, and Exocytosis via RAB11-positive Carriers

Cited by 123 publications

References 51 publications

The Single Dynamin-like Protein of Trypanosoma brucei Regulates Mitochondrial Division and Is Not Required for Endocytosis

The Single Dynamin-like Protein of Trypanosoma brucei Regulates Mitochondrial Division and Is Not Required for Endocytosis

Factors Affecting the Level and Localization of the Transferrin Receptor in Trypanosoma brucei

Membrane domains and flagellar pocket boundaries are influenced by the cytoskeleton in African trypanosomes

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