Ubiquitylation is Required for Degradation of Transmembrane Surface Proteins in Trypanosomes

Chung, Wei-Lian; Leung, Ka Fai; Carrington, Mark; Field, Mark C.

doi:10.1111/j.1600-0854.2008.00785.x

Cited by 59 publications

(98 citation statements)

References 58 publications

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“…An in trans decrease in expression levels of ESCRT I (Vps23 and Vps28) and retromer complex (Vps26) proteins on Rab28 knockdown suggests a functional connection between Rab28, ESCRT and retromer. The effects on ESCRT are fully consistent with the observed partial protection of ISG65 and ISG75, as both require ubiquitylation for turnover and, hence, are probably sorted by the ESCRT system (Chung et al, 2008;Leung et al, 2011). The effects on the Rab11 recycling compartment suggest increased cycling of cargo between endosomes, as reported upon silencing of Vps26 and Vps23 in mammalian cells (Seaman, 2004;Razi and Futter, 2006;Raiborg et al, 2008).…”

Section: Discussionsupporting

confidence: 73%

“…We were unable to detect any significant change in ISG65 or ISG75 levels on the surface of the cells by FACS analysis, but as we did not have an accurate estimate of relative internal and external pool sizes, this might reflect changes in a comparatively small intracellular pool (data not shown). Previous analysis demonstrated that, in common with higher eukaryotes, turnover of ubiquitylated proteins is dependent on the ESCRT pathway (Chung et al, 2008), so we propose that the increase in intracellular ISG65 and ISG75 following Rab28 depletion is due to failure to progress to the lysosome, similar to the effects of Rab28 RNAi on both ConA and TLF.…”

Section: Rab28 Function In Endocytosis 3775mentioning

confidence: 60%

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Rab28 function in trypanosomes: interactions with retromer and ESCRT pathways

Lumb

Leung

DuBois

et al. 2011

Journal of Cell Science

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SummaryEarly endosomal cargo is typically targeted to either a degradative or recycling pathway. Despite established functions for the retromer and ESCRT complexes at late endosomes/multivesicular bodies, the mechanisms integrating and coordinating these functions remain largely unknown. Rab family GTPases are key membrane trafficking organizers and could contribute. Here, in the unicellular organism Trypanosoma brucei, we demonstrate that Rab28 locates to the endosomal pathway and partially colocalizes with Vps23, an ESCRT I component. Rab28 is required for turnover of endocytosed proteins and for lysosomal delivery of protein cargo. Using RNA interference we find that in Rab28-depleted cells, protein levels of ESCRT I (Vps23/28) and retromer (Vps26) are also decreased, suggesting that Rab28 is an important regulator of these factors. We suggest that Rab28 coordinates the activity of retromer-dependent trafficking and ESCRT-mediated degradative pathways.

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Section: Discussionsupporting

confidence: 73%

Section: Rab28 Function In Endocytosis 3775mentioning

confidence: 60%

Rab28 function in trypanosomes: interactions with retromer and ESCRT pathways

Lumb

Leung

DuBois

et al. 2011

Journal of Cell Science

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“…However, we previously found that ISG65 is present on both cell surface and endosomal membranes and turned over significantly more rapidly than VSG, suggesting differential sorting (7). More recently, we demonstrated that lysine residues in the ISG65 cytoplasmic domain are important for regulating trafficking and stability in vivo (8). Furthermore, we showed that this was mediated by a ubiquitin-and ESCRTdependent mechanism (8,25).…”

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confidence: 82%

Ubiquitylation and Developmental Regulation of Invariant Surface Protein Expression in Trypanosomes

et al. 2011

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The cell surface of Trypanosoma brucei is dominated by the glycosylphosphatidylinositol-anchored variant surface glycoprotein (VSG), which is essential for immune evasion. VSG biosynthesis, trafficking, and turnover are well documented, but trans-membrane domain (TMD) proteins, including the invariant surface glycoproteins (ISGs), are less well characterized. Internalization and degradation of ISG65 depend on ubiquitylation of conserved cytoplasmic lysines. Using epitope-tagged ISG75 and reporter chimeric proteins bearing the cytoplasmic and trans-membrane regions of ISG75, together with multiple mutants with lysine-to-arginine mutations, we demonstrate that the cytoplasmic tail of ISG75 is both sufficient and necessary for endosomal targeting and degradation. The ISG75 chimeric reporter protein localized to endocytic organelles, while lysine-null versions were significantly stabilized at the cell surface. Importantly, ISG75 cytoplasmic lysines are modified by extensive oligoubiquitin chains and ubiquitylation is abolished in the lysine-null version. Furthermore, we find evidence for differential modes of turnover of ISG65 and ISG75. Full-length lysine-null ISG65 localization and protein turnover are significantly perturbed, but ISG75 localization and protein turnover are not, while ubiquitin conjugates can be detected for full-length lysine-null ISG75 but not ISG65. We find that the ISG75 ectodomain has a predicted coiled-coil, suggesting that ISG75 could be part of a complex, while ISG65 behaves independently. We also demonstrate a developmental stage-specific mechanism for exclusion of surface ISG expression in insect-stage cells by a ubiquitin-independent mechanism. We suggest that ubiquitylation may be a general mechanism for regulating trans-membrane domain surface proteins in trypanosomes.In higher eukaryotes, internalization and sorting of surface trans-membrane domain (TMD) proteins require specific signals within the cytoplasmic domain that are recognized by multiple factors within the endocytic machinery (5). One pathway involves covalent attachment of ubiquitin to substrate lysine residues within the cytoplasmic domain of TMD proteins. Ubiquitylation is mediated by an enzymatic cascade terminating in an E3 ubiquitin ligase, which transfers thioesterlinked ubiquitin to the substrate lysine-NH 2 group, forming an isopeptide bond (23).

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“…Furthermore, we found that RNAi knockdown of T. brucei Vps5 resulted in a modest but significant decrease to intracellular levels of ISG75 (Fig. 11C), a transmembrane protein that traffics through the endosomal system and undergoes ubiquitin-dependent degradation (Chung et al, 2008;Leung et al, 2008). The decrease in ISG75 endosome-associated staining is at least in part due to accelerated turnover, and cannot simply be attributed to defective uptake, as T. brucei Vps5 RNAi 1502 Journal of Cell Science 124 (9) leads to more rapid degradation (Fig.…”

Section: Localisation and Functional Analysis Of The Retromer Complexmentioning

confidence: 98%

Evolutionary reconstruction of the retromer complex and its function in Trypanosoma brucei

Koumandou

Klute

Herman

et al. 2011

Journal of Cell Science

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SummaryIntracellular trafficking and protein sorting are mediated by various protein complexes, with the retromer complex being primarily involved in retrograde traffic from the endosome or lysosome to the Golgi complex. Here, comparative genomics, cell biology and phylogenetics were used to probe the early evolution of retromer and its function. Retromer subunits Vps26, Vps29 and Vps35 are near universal, and, by inference, the complex was an ancient feature of eukaryotic cells. Surprisingly, we found DSCR3, a Vps26 paralogue in humans associated with Down's syndrome, in at least four eukaryotic supergroups, implying a more ancient origin than previously suspected. By contrast, retromer cargo proteins showed considerable interlineage variability, with lineage-specific and broadly conserved examples found. Vps10 trafficking probably represents an ancestral role for the complex. Vps5, the BAR-domaincontaining membrane-deformation subunit, was found in diverse eukaryotes, including in the divergent eukaryote Trypanosoma brucei, where it is the first example of a BAR-domain protein. To determine functional conservation, an initial characterisation of retromer was performed in T. brucei; the endosomal localisation and its role in endosomal targeting are conserved. Therefore retromer is identified as a further feature of the sophisticated intracellular trafficking machinery of the last eukaryotic common ancestor, with BAR domains representing a possible third independent mechanism of membrane-deformation arising in early eukaryotes.

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Ubiquitylation is Required for Degradation of Transmembrane Surface Proteins in Trypanosomes

Cited by 59 publications

References 58 publications

Rab28 function in trypanosomes: interactions with retromer and ESCRT pathways

Rab28 function in trypanosomes: interactions with retromer and ESCRT pathways

Ubiquitylation and Developmental Regulation of Invariant Surface Protein Expression in Trypanosomes

Evolutionary reconstruction of the retromer complex and its function in Trypanosoma brucei

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