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. 2015 Oct 1;471(1):79-88.
doi: 10.1042/BJ20150336. Epub 2015 Jul 28.

A non-canonical ESCRT pathway, including histidine domain phosphotyrosine phosphatase (HD-PTP), is used for down-regulation of virally ubiquitinated MHC class I

Michael D J Parkinson  1 , Siân C Piper  1 , Nicholas A Bright  1 , Jennifer L Evans  1 , Jessica M Boname  2 , Katherine Bowers  1 , Paul J Lehner  2 , J Paul Luzio  3
Affiliations

A non-canonical ESCRT pathway, including histidine domain phosphotyrosine phosphatase (HD-PTP), is used for down-regulation of virally ubiquitinated MHC class I

Michael D J Parkinson et al. Biochem J. .

Abstract

The Kaposi's sarcoma-associated herpes virus (KSHV) K3 viral gene product effectively down-regulates cell surface MHC class I. K3 is an E3 ubiquitin ligase that promotes Lys(63)-linked polyubiquitination of MHC class I, providing the signal for clathrin-mediated endocytosis. Endocytosis is followed by sorting into the intralumenal vesicles (ILVs) of multivesicular bodies (MVBs) and eventual delivery to lysosomes. The sorting of MHC class I into MVBs requires many individual proteins of the four endosomal sorting complexes required for transport (ESCRTs). In HeLa cells expressing the KSHV K3 ubiquitin ligase, the effect of RNAi-mediated depletion of individual proteins of the ESCRT-0 and ESCRT-I complexes and three ESCRT-III proteins showed that these are required to down-regulate MHC class I. However, depletion of proteins of the ESCRT-II complex or of the ESCRT-III protein, VPS20 (vacuolar protein sorting 20)/CHMP6 (charged MVB protein 6), failed to prevent the loss of MHC class I from the cell surface. Depletion of histidine domain phosphotyrosine phosphatase (HD-PTP) resulted in an increase in the cell surface concentration of MHC class I in HeLa cells expressing the KSHV K3 ubiquitin ligase. Rescue experiments with wild-type (WT) and mutant HD-PTP supported the conclusion that HD-PTP acts as an alternative to ESCRT-II and VPS20/CHMP6 as a link between the ESCRT-I and those ESCRT-III protein(s) necessary for ILV formation. Thus, the down-regulation of cell surface MHC class I, polyubiquitinated by the KSHV K3 ubiquitin ligase, does not employ the canonical ESCRT pathway, but instead utilizes an alternative pathway in which HD-PTP replaces ESCRT-II and VPS20/CHMP6.

Keywords: endocytosis; endosomal sorting complex required for transport (ESCRT); histidine domain phosphotyrosine phosphatase (HD-PTP); histidine-domain protein tyrosine phosphatase non-receptor type 23 (PTPN23); lysosome; ubiquitination (ubiquitylation).

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Figures

Figure 1
Figure 1. MHC class I molecules from the cell surface of HeLa-KK3 cells are endocytosed into MVBs but not the TGN
(A) HeLa-KK3 cells treated with IFNγ and incubated with HRP-conjugated anti-MHC class I for 90 min at 37°C were fixed, incubated with DAB/H2O2 and processed for EM. Endocytosed HRP-labelled anti-MHC class I antibodies accumulated in MVBs (arrowheads), as revealed by the electron dense HRP/DAB reaction product. Scale bar, 1 μm. (B) HeLa-KK3 cells were treated with IFNγ, incubated with FITC-conjugated anti-MHC class I for 90 min at 37°C and frozen ultrathin sections were immunolabelled with anti-FITC (15 nm gold). A representative MVB is shown containing accumulated endocytosed anti-MHC class I antibodies (arrowhead). Scale bar, 200 nm. (C) Quantification of immunogold labelled FITC-conjugated anti-MHC class I on frozen ultrathin sections after uptake for 90 min at 37°C. Mean ± S.E.M. of gold particles counted. Abbreviation: OMA, other membrane-associated gold particles.
Figure 2
Figure 2. The effect of depleting ESCRT proteins on down-regulation of virally-ubiquitinated MHC class I
(A) Summary of cytofluorometric analysis of cell surface expression of MHC class I in HeLa-KK3 cells after depletion of individual ESCRT proteins with siRNA pools. In each experiment geometric mean fluorescence data from siRNA-treated cells were compared with data from mock-treated cells, normalized to 100%. Mean ± S.E.M., number of experiments in brackets, P-values **≤0.01, *<0.05. (B–G) Representative cytofluorometry traces of cell surface expression of MHC class I in HeLa-KK3 cells following depletion of individual ESCRT proteins (unfilled black traces). Traces for secondary antibody controls (filled black traces), mock-treated HeLa-KK3 (unfilled grey traces) and HeLa (filled grey traces) cells are also shown. The effectiveness of knockdown is shown by either immunoblotting or real time quantitative PCR (mean ± S.E.M. of three samples). CALR, calreticulin. (H) Pulse-chase analysis of degradation of MHC class I HeLa-KK3 cells following depletion of individual ESCRT proteins. The immunoprecipitation of CANX (calnexin), with mouse anti-CANX antibody AF8 from the pulse-chase radiolabelled cell lysates provided the input lysate control.
Figure 3
Figure 3. The effect of depleting VPS20/CHMP6 on endocytic compartments
(A) Immunofluorescence confocal microscopy of HeLa cells stained with anti-ubiquitin antibodies, following mock knockdown or knockdown with either a pool of siRNAs or a single siRNA, oligo1 (upper panels). Lower panels show stably-transfected HeLa cells expressing oligo1 siRNA-resistant myc-tagged VPS20 (myc-VPS20RNAires HeLa cells), after the same knockdown treatments. Scale bar=10 μm (B) Immunoblotting to show expression of VPS20 and myc-tagged VPS20 in the HeLa cells and myc-VPS20RNAires HeLa cells following siRNA knockdown. *myc-VPS20; CALR, calreticulin. (C) Proportion of cells with enlarged, ubiquitinated compartment phenotype in HeLa cells or myc-VPS20RNAires HeLa cells after depletion of endogenous VPS20 with either a pool of siRNAs or oligo1. Mean ± S.E.M. (three coverslips, 50 cells per coverslip). (D) Transmission electron micrograph of a HeLa cell treated with the VPS20 siRNA pool, showing a swollen endocytic compartment and associated MVBs. Scale bar=500nm. (E) Transmission electron micrograph of an anti-FITC antibody (15 nm gold)-stained frozen section from a HeLa-KK3 cell allowed to endocytose FITC-conjugated anti-MHC class I for 90 min at 37°C after knockdown of VPS20. Two representative MVBs are shown containing accumulated endocytosed anti-MHC class I antibodies. Scale bar=200nm.
Figure 4
Figure 4. The effect of depleting HD-PTP on down-regulation of virally ubiquitinated MHC class I
(A) Summary of cytofluorometric analysis of cell surface expression of MHC class I in HeLa-KK3 cells following mock knockdown or depletion of HD-PTP with an siRNA pool. Geometric mean fluorescence data from siRNA-treated cells were compared with data from mock-treated cells, normalized to 100%. Mean ± S.E.M., number of experiments in brackets, P-value **≤0.01. (B–D) Representative cytofluorometry traces of cell surface expression of MHC class I in HeLa-KK3 cells following depletion of HD-PTP (unfilled black traces) with an siRNA pool (B) or individual siRNAs from the pool, oligo3 (C) and oligo4 (D). Traces for mock treated HeLa-KK3 cells (unfilled grey traces) and secondary antibody controls (filled black traces) are also shown. (E) Representative cytofluorometry trace of cell surface expression of MHC class I in HeLa cells following depletion of HD-PTP with a siRNA pool (unfilled black trace). Traces for mock-treated HeLa cells (filled grey trace) and secondary antibody control (filled black trace) are also shown. (F) Real time quantitative PCR to show the effectiveness of knockdown in the representative experiments shown in panels (B–E). Mean ± S.E.M. of three samples.
Figure 5
Figure 5. Rescuing the down-regulation of virally ubiquitinated MHC class I following knockdown of HD-PTP
(A) HeLa-KK3 cells were treated with either non-targeting siRNA (NT) or HD-PTP oligo2 siRNA on day 1 and then 12 h later transfected with pEGFP-C3 plasmids containing inserts encoding WT, oligo2 siRNA resistant WT (WTRNAires) or oligo2 siRNA resistant L202D/I206D mutant (MutRNAires) GFP-HD-PTP. On day 4, the presence of GFP-HD-PTP in harvested cells was detected by immunoblotting with anti-GFP. (B–D) Representative cytofluorometry traces of cell surface expression of MHC class I in HeLa-KK3 cells treated with oligo2 siRNA and transfected with GFP-HD-PTP constructs (unfilled black traces). In (B and C), cells were treated with oligo2 siRNA and transfected with WT GFP-HD-PTPRNAires before analysing the GFP-positive (B) and negative (C) cells. In (D), cells were treated with oligo2 siRNA and transfected with WT GFP-HD-PTP and, in (E), treated with oligo2 siRNA and transfected with L202D/I206D mutated Mut GFP-HD-PTPRNAires. Traces for secondary antibody controls (filled black traces) and mock-treated HeLa-KK3 (unfilled grey traces) are also shown.
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References

    1. Boname J.M., Lehner P.J. What has the study of the K3 and K5 viral ubiquitin E3 ligases taught us about ubiquitin-mediated receptor regulation? Viruses. 2011;3:118–131. doi: 10.3390/v3020118. - DOI - PMC - PubMed
    1. Lehner P.J., Hoer S., Dodd R., Duncan L.M. Downregulation of cell surface receptors by the K3 family of viral and cellular ubiquitin E3 ligases. Immunol. Rev. 2005;207:112–125. doi: 10.1111/j.0105-2896.2005.00314.x. - DOI - PubMed
    1. Burr M.L., van den Boomen D.J., Bye H., Antrobus R., Wiertz E.J., Lehner P.J. MHC class I molecules are preferentially ubiquitinated on endoplasmic reticulum luminal residues during HRD1 ubiquitin E3 ligase-mediated dislocation. Proc. Natl. Acad. Sci. U.S.A. 2013;110:14290–14295. doi: 10.1073/pnas.1303380110. - DOI - PMC - PubMed
    1. Duncan L.M., Piper S., Dodd R.B., Saville M.K., Sanderson C.M., Luzio J.P., Lehner P.J. Lysine-63-linked ubiquitination is required for endolysosomal degradation of class I molecules. EMBO J. 2006;25:1635–1645. doi: 10.1038/sj.emboj.7601056. - DOI - PMC - PubMed
    1. Hewitt E.W., Duncan L., Mufti D., Baker J., Stevenson P.G., Lehner P.J. Ubiquitylation of MHC class I by the K3 viral protein signals internalization and TSG101-dependent degradation. EMBO J. 2002;21:2418–2429. doi: 10.1093/emboj/21.10.2418. - DOI - PMC - PubMed

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