Ubiquitin chain restriction (UbiCRest) analysis reveals K63, K27, and K29 ubiquitin chain association with CTLA4-HA. (A) Representative UbiCRest analysis of western blots for CTLA4-HA. HeLa S3 Flp-In CTLA4-HA were transfected for 72 h with non-targeting (NT1) or USP8 siRNA prior to lysis and HA-immunoprecipitation (IP; IB: Immunoblot). CTLA4-HA-beads were treated with indicated DUBs or buffer only (Mock) for 1 h at 37°C, and analyzed alongside supernatants (released ubiquitin species). Specific activities of DUBs as reported in the literature are depicted in the key above the blot. Ub-P indicates the ability to cleave proximal ubiquitin; S/T denotes activity for serine or threonine linkages. All l.s.: All linkage-specific DUBs (OTULIN, OTUB1*, AMSH*, LotAN, Cezanne, TRABID, OTUD2, TssM*). (B) Quantification of Ub-CTLA4-HA signal remaining after DUB-treatment shown relative to Mock (“buffer only” control) for data represented in A. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (C) Representative western blots of CTLA4-HA immunoprecipitated (IP) under denaturing conditions were probed with K63 (top), K27 (middle), and K29 (bottom) ubiquitin chain linkage-specific antibodies. (D) Left Panel: A balance of E3 ligase and deubiquitylating activity governs receptor fate at the endosome. Endosomal USP8 is proposed to recycle ubiquitin from CTLA4 prior as well as after commitment to the lysosomal degradation pathway. Middle Panel: In the absence of USP8, a complex pattern of ubiquitylation accrues, composed of conventional Lys63 and unusual Lys27 and Lys29 ubiquitin linkages. This would normally lead to rapid degradation but is countermanded by downstream effects of USP8 loss that inhibit delivery to a degradative lysosome. That some ubiquitylated CTLA4 reaches the internal vesicles of the multivesicular body can be inferred from its presence in exosomes released upon v-ATPase inhibition. Right panel: Mutation of all lysines (K-null) or just the critical two lysines (K203 and K213) to arginines in the cytoplasmic tail of CTLA4 interferes with endolysosomal sorting and downregulation. These CTLA4 mutants accumulate in early (sorting) endosomes and fail to progress to later endolysosomal compartments. For reasons of clarity, mono-ubiquitylation, branching, and diversity of chain length are not depicted. Source data are available for this figure: SourceData F10.
Figure 10.

Ubiquitin chain restriction (UbiCRest) analysis reveals K63, K27, and K29 ubiquitin chain association with CTLA4-HA. (A) Representative UbiCRest analysis of western blots for CTLA4-HA. HeLa S3 Flp-In CTLA4-HA were transfected for 72 h with non-targeting (NT1) or USP8 siRNA prior to lysis and HA-immunoprecipitation (IP; IB: Immunoblot). CTLA4-HA-beads were treated with indicated DUBs or buffer only (Mock) for 1 h at 37°C, and analyzed alongside supernatants (released ubiquitin species). Specific activities of DUBs as reported in the literature are depicted in the key above the blot. Ub-P indicates the ability to cleave proximal ubiquitin; S/T denotes activity for serine or threonine linkages. All l.s.: All linkage-specific DUBs (OTULIN, OTUB1*, AMSH*, LotAN, Cezanne, TRABID, OTUD2, TssM*). (B) Quantification of Ub-CTLA4-HA signal remaining after DUB-treatment shown relative to Mock (“buffer only” control) for data represented in A. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (C) Representative western blots of CTLA4-HA immunoprecipitated (IP) under denaturing conditions were probed with K63 (top), K27 (middle), and K29 (bottom) ubiquitin chain linkage-specific antibodies. (D) Left Panel: A balance of E3 ligase and deubiquitylating activity governs receptor fate at the endosome. Endosomal USP8 is proposed to recycle ubiquitin from CTLA4 prior as well as after commitment to the lysosomal degradation pathway. Middle Panel: In the absence of USP8, a complex pattern of ubiquitylation accrues, composed of conventional Lys63 and unusual Lys27 and Lys29 ubiquitin linkages. This would normally lead to rapid degradation but is countermanded by downstream effects of USP8 loss that inhibit delivery to a degradative lysosome. That some ubiquitylated CTLA4 reaches the internal vesicles of the multivesicular body can be inferred from its presence in exosomes released upon v-ATPase inhibition. Right panel: Mutation of all lysines (K-null) or just the critical two lysines (K203 and K213) to arginines in the cytoplasmic tail of CTLA4 interferes with endolysosomal sorting and downregulation. These CTLA4 mutants accumulate in early (sorting) endosomes and fail to progress to later endolysosomal compartments. For reasons of clarity, mono-ubiquitylation, branching, and diversity of chain length are not depicted. Source data are available for this figure: SourceData F10.

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