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. 2010 Feb;11(2):210-20.
doi: 10.1111/j.1600-0854.2009.01011.x. Epub 2009 Nov 17.

Efficient internalization of MHC I requires lysine-11 and lysine-63 mixed linkage polyubiquitin chains

Jessica M Boname  1 , Mair Thomas, Helen R Stagg, Ping Xu, Junmin Peng, Paul J Lehner
Affiliations
Free PMC article

Efficient internalization of MHC I requires lysine-11 and lysine-63 mixed linkage polyubiquitin chains

Jessica M Boname et al. Traffic. 2010 Feb.
Free PMC article

Abstract

The downregulation of cell surface receptors by endocytosis is a fundamental requirement for the termination of signalling responses and ubiquitination is a critical regulatory step in receptor regulation. The K5 gene product of Kaposi's sarcoma-associated herpesvirus is an E3 ligase that ubiquitinates and downregulates several cell surface immunoreceptors, including major histocompatibility complex (MHC) class I molecules. Here, we show that K5 targets the membrane proximal lysine of MHC I for conjugation with mixed linkage polyubiquitin chains. Quantitative mass spectrometry revealed an increase in lysine-11, as well as lysine-63, linked polyubiquitin chains on MHC I in K5-expressing cells. Using a combination of mutant ubiquitins and MHC I molecules expressing a single cytosolic lysine residue, we confirm a functional role for lysines-11 and -63 in K5-mediated MHC I endocytosis. We show that lysine-11 linkages are important for receptor endocytosis, and that complex mixed linkage polyubiquitin chains are generated in vivo.

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Figures

Figure 1
Figure 1
K5 polyubiquitinates the membrane proximal cytosolic tail lysine residue of MHC I. A) K3 downregulates MHC I more effectively than K5. Cytofluorometric analysis of MHC I expression in HeLa, HeLa-K3, HeLa-K5 or Cy5 control stained cells. B) K5 causes a predominantly monoubiquitinated MHC I species. Radiolabelled HeLa, HeLa-K3 or HeLa-K5 cells were washed and stained with w6/32 to detect only cell surface MHC I prior to detergent lysis. Postlysis samples were split and processed to detect either surface (S) or total (T) MHC I by w6/32 primary IP and HC10 reIP. C) K3 and K5 increase MHC I internalization in HeLa cells. MHC I internalization was determined in a cytofluorometric-based internalization assay. The change in mean fluorescence intensity (MFI) of the w6/32-reactive MHC I remaining at the cell surface was plotted as a function of time (mean of four replicates ± SEM). D) The cytosolic tail of the wild-type MHC I molecule HLA*A0201 (A2KKK) and mutants (lysine residues in bold). E) Cytofluorometric analysis of K5's effects on HLA-A2 and mutants. HeLa cell lines expressing wt and mutant HLA-A2 were transduced with a lentivirus expressing K5 and GFP. Cells were stained with the HLA-A2-specific BB7.2 mAb. Untransduced cells (solid lines), K5 transduced (grey filled) and Cy5 control staining (black filled). The fold downregulation of HLA-A2 by K5 is noted above each histogram. F) HeLa-A2 expressing cell lines ± K5 were radiolabelled and lysed followed by primary IP with BB7.2 and reIP with MR24.
Figure 2
Figure 2
Polyubiquitination of MHC I by K5 requires ubcH5 and ubc13. MHC I expression in HeLa-K5 cells is rescued following cellular depletion of ubcH5c and ubc13. A) Cytofluorometric analysis of class I expression 60 h postdepletion of ubcH5c or ubc13 by siRNA. HeLa-K5 + mock siRNA (black line), HeLa (dotted line) HeLa-K5 + siRNA for either ubcH5c or ubc13 (grey filled) and Cy5 control (black filled). B) Immunoblot confirms the siRNA depletion of ubcH5c and ubc13. C) HeLa-K5 cells from (A), depleted of ubcH5c or ubc13, were radiolabelled, lysed and subjected to w6/32 primary IP and HC10 reIP.
Figure 3
Figure 3
Quantitative MS identifies Lys11 and Lys63 ubiquitin chain linkages on MHC I from HeLa-K5 cells. A) Isolated membrane fractions from HeLa and HeLa-K5 cells were solubilized and denatured class I heavy chains were immunoprecipitated with HC10. Following SDS–PAGE and Coomasie staining, the left half of the gel (containing radiolabelled samples) was dried and analysed by phosphorimager. Gel slices (as labelled #1–6), from the right half, were separated, digested and analysed by MS. B) Amino acid sequence of the HLA-A28 class I allele showing peptides retrieved from MS analysis following MHC IIP. Also indicated are the signal sequence, transmembrane domain and lysine 335 (K), the dominant ubiquitin acceptor of K5-mediated polyubiquitination. C) Quantification of ubiquitin chain linkages attached to MHC I by the MS-based AQUA method. Peptides were eluted from gel slices #3 and #5 from HeLa-K5 and #2 and #4 from HeLa and quantitated with the addition of eight stable isotope-labelled peptides as internal standards, including all seven –GG peptides and one ubiquitin peptide (Thr55-Lys63). D) Graphical representation of the data in part (C).
Figure 4
Figure 4
Lys11 and Lys63 of ubiquitin are necessary for K5-mediated MHC I endocytosis. A) K5 downregulation of HLA-A2KRA (containing the single cytosolic lysine K335) is rescued by overexpression of the ubiquitin mutants R6, R11 or R63. Cytofluorometric analysis of HLA-A2KRA expression in K 5+ cells 3 days post-transduction with ubiquitin mutants as noted. Cells were stained with BB7.2 + Cy5. B) Overexpression of R11 and R63, but not R6 or R48 ubiquitin mutants, interferes with K5-mediated MHC I endocytosis. Internalization of MHC I in HeLa-K5 cells transduced with wt or mutant Ub-GFP 3 days post-transduction as noted. Cells were assayed as in Figure 1C and cells transduced with lentivirus were gated for GFPhigh expression prior to analysis. Mean of three replicates ± SEM.
Figure 5
Figure 5
Lys6, Lys11 and Lys63 of ubiquitin are necessary and sufficient for K5-mediated MHC I endocytosis. A) K5 downregulation of MHC I with a single cytosolic lysine (K335) is restored by overexpression of a ubiquitin mutant expressing only three lysine residues (K6/11/63). Cytofluorometric analysis of HLA-A2KRA expression in K 5+ cells 3 days post-transduction with ubiquitin mutants as noted. Cells were stained with BB7.2 + Cy5. B) Internalization assays (as in Figure 4B) of HeLa-K5 cells transduced with the KoUb-GFP-based mutants as noted. Mean of four replicates ± SEM.
Figure 6
Figure 6
Potential configuration of mixed linkage Lys11- and Lys63-linked polyubiquitin chains. A) Mixed linkage chains can consist of non-homogeneous chains or forked chains as depicted. B) HeLa-K5 cells were transduced with a mixture of the K6/11 ubiquitin mutant to a GFP level sufficient to see a rescue of cell surface MHC I, and increasing concentrations of the K6/63 ubiquitin mutant. Conversely, starting with the K6/63 mutant and titrating in the K6/11 mutant also failed to restore K5 function, while the control K6/11/63 ubiquitin mutant fully supported K5-mediated downregulation of MHC I.
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