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Ubiquitination of human leukocyte antigen (HLA)-DM by different membrane-associated RING-CH (MARCH) protein family E3 ligases targets different endocytic pathways.

Jahnke M, Trowsdale J, Kelly AP - J. Biol. Chem. (2012)

Bottom Line: Here, we show that DM also undergoes post-translational modification through ubiquitination of a single lysine residue present in the cytoplasmic tail of the α chain, DMα.In contrast, MARCH8-induced loss of surface DM was entirely dependent upon the tyrosine signal on DMβ.The influence of MARCH8 was indirect and may have resulted from modification of components of the endocytic machinery by ubiquitination.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom.

ABSTRACT
HLA-DM plays an essential role in the peptide loading of classical class II molecules and is present both at the cell surface and in late endosomal peptide-loading compartments. Trafficking of DM within antigen-presenting cells is complex and is, in part, controlled by a tyrosine-based targeting signal present in the cytoplasmic tail of DMβ. Here, we show that DM also undergoes post-translational modification through ubiquitination of a single lysine residue present in the cytoplasmic tail of the α chain, DMα. Ubiquitination of DM by MARCH1 and MARCH9 induced loss of DM molecules from the cell surface by a mechanism that cumulatively involved both direct attachment of ubiquitin chains to DMα and a functional tyrosine-based signal on DMβ. In contrast, MARCH8-induced loss of surface DM was entirely dependent upon the tyrosine signal on DMβ. In the absence of this tyrosine residue, levels of DM remained unchanged irrespective of whether DMα was ubiquitinated by MARCH8. The influence of MARCH8 was indirect and may have resulted from modification of components of the endocytic machinery by ubiquitination.

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MARCH E3 ligase down-regulation of DM is influenced by a YxxΦ endocytosis motif and DMα ubiquitination. HEK-293T cells were transiently transfected with DMα and DMβ wild-type or mutant constructs and either EGFP-MARCH1, 8, or 9. Cell surface HLA-DM expression was determined by FACS analysis after staining with anti-DM antibody MaP.DM1 and PE anti-mouse mAb. Surface expression was calculated as: % DM surface expression = (MFI of cells transfected with EGFP-MARCH × 100)/(MFI of cells transfected with EGFP vector alone). Data from independent experiments are shown by different shapes; means are represented by black bars. The corresponding FACS plots are shown in supplemental Fig. 2.
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Figure 6: MARCH E3 ligase down-regulation of DM is influenced by a YxxΦ endocytosis motif and DMα ubiquitination. HEK-293T cells were transiently transfected with DMα and DMβ wild-type or mutant constructs and either EGFP-MARCH1, 8, or 9. Cell surface HLA-DM expression was determined by FACS analysis after staining with anti-DM antibody MaP.DM1 and PE anti-mouse mAb. Surface expression was calculated as: % DM surface expression = (MFI of cells transfected with EGFP-MARCH × 100)/(MFI of cells transfected with EGFP vector alone). Data from independent experiments are shown by different shapes; means are represented by black bars. The corresponding FACS plots are shown in supplemental Fig. 2.

Mentions: The molecular basis of MARCH E3 ligase target specificity is poorly understood (31, 32). To compare the interaction of MARCH1, 8, and 9 with DM, we monitored changes in DM expression in transiently transfected 293T cells (Fig. 6). MARCH1 down-regulated DM most efficiently (21% surface expression remaining in the presence of MARCH1 compared with EGFP control) followed by MARCH9 (28%) and then MARCH8 (49%).


Ubiquitination of human leukocyte antigen (HLA)-DM by different membrane-associated RING-CH (MARCH) protein family E3 ligases targets different endocytic pathways.

Jahnke M, Trowsdale J, Kelly AP - J. Biol. Chem. (2012)

MARCH E3 ligase down-regulation of DM is influenced by a YxxΦ endocytosis motif and DMα ubiquitination. HEK-293T cells were transiently transfected with DMα and DMβ wild-type or mutant constructs and either EGFP-MARCH1, 8, or 9. Cell surface HLA-DM expression was determined by FACS analysis after staining with anti-DM antibody MaP.DM1 and PE anti-mouse mAb. Surface expression was calculated as: % DM surface expression = (MFI of cells transfected with EGFP-MARCH × 100)/(MFI of cells transfected with EGFP vector alone). Data from independent experiments are shown by different shapes; means are represented by black bars. The corresponding FACS plots are shown in supplemental Fig. 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3293585&req=5

Figure 6: MARCH E3 ligase down-regulation of DM is influenced by a YxxΦ endocytosis motif and DMα ubiquitination. HEK-293T cells were transiently transfected with DMα and DMβ wild-type or mutant constructs and either EGFP-MARCH1, 8, or 9. Cell surface HLA-DM expression was determined by FACS analysis after staining with anti-DM antibody MaP.DM1 and PE anti-mouse mAb. Surface expression was calculated as: % DM surface expression = (MFI of cells transfected with EGFP-MARCH × 100)/(MFI of cells transfected with EGFP vector alone). Data from independent experiments are shown by different shapes; means are represented by black bars. The corresponding FACS plots are shown in supplemental Fig. 2.
Mentions: The molecular basis of MARCH E3 ligase target specificity is poorly understood (31, 32). To compare the interaction of MARCH1, 8, and 9 with DM, we monitored changes in DM expression in transiently transfected 293T cells (Fig. 6). MARCH1 down-regulated DM most efficiently (21% surface expression remaining in the presence of MARCH1 compared with EGFP control) followed by MARCH9 (28%) and then MARCH8 (49%).

Bottom Line: Here, we show that DM also undergoes post-translational modification through ubiquitination of a single lysine residue present in the cytoplasmic tail of the α chain, DMα.In contrast, MARCH8-induced loss of surface DM was entirely dependent upon the tyrosine signal on DMβ.The influence of MARCH8 was indirect and may have resulted from modification of components of the endocytic machinery by ubiquitination.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom.

ABSTRACT
HLA-DM plays an essential role in the peptide loading of classical class II molecules and is present both at the cell surface and in late endosomal peptide-loading compartments. Trafficking of DM within antigen-presenting cells is complex and is, in part, controlled by a tyrosine-based targeting signal present in the cytoplasmic tail of DMβ. Here, we show that DM also undergoes post-translational modification through ubiquitination of a single lysine residue present in the cytoplasmic tail of the α chain, DMα. Ubiquitination of DM by MARCH1 and MARCH9 induced loss of DM molecules from the cell surface by a mechanism that cumulatively involved both direct attachment of ubiquitin chains to DMα and a functional tyrosine-based signal on DMβ. In contrast, MARCH8-induced loss of surface DM was entirely dependent upon the tyrosine signal on DMβ. In the absence of this tyrosine residue, levels of DM remained unchanged irrespective of whether DMα was ubiquitinated by MARCH8. The influence of MARCH8 was indirect and may have resulted from modification of components of the endocytic machinery by ubiquitination.

Show MeSH
Related in: MedlinePlus