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A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition.

Coscoy L, Sanchez DJ, Ganem D - J. Cell Biol. (2001)

Bottom Line: Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells.This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins.Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Microbiology, University of California Medical Center, San Francisco, CA 94143, USA.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells. This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins. Here, we show that expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface. MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity. In vitro, addition of a MIR2-glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion; this reaction is abolished by mutation of the Zn-coordinating residues of the PHD domain. Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

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Addition of lysine into B7.1 enable its downregulation. (A) Sequence of the TM and intracytoplasmic regions of B7.1 (top line) and B7.1KKK mutant (bottom line). The mutant residues within the cytoplasmic region of B7.1KKK are indicated in boldface. (B) B7.1 wt and KKK mutant constructs were stably expressed in HeLa cells. Cells were transduced a second time by pBP-MIR2/EGFP, and surface expression of the B7.1 molecules was analyzed by flow cytometry. (C) Cells expressing B7.1 wt or KKK mutant proteins were stably transduced by pMX-pie, pBP-MIR1/EGFP, or pBP-MIR2/EGFP. From each set of transductants, B7.1 molecules were immunoprecipitated and analyzed by Western blot using an anti-Ub antibody.
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fig5: Addition of lysine into B7.1 enable its downregulation. (A) Sequence of the TM and intracytoplasmic regions of B7.1 (top line) and B7.1KKK mutant (bottom line). The mutant residues within the cytoplasmic region of B7.1KKK are indicated in boldface. (B) B7.1 wt and KKK mutant constructs were stably expressed in HeLa cells. Cells were transduced a second time by pBP-MIR2/EGFP, and surface expression of the B7.1 molecules was analyzed by flow cytometry. (C) Cells expressing B7.1 wt or KKK mutant proteins were stably transduced by pMX-pie, pBP-MIR1/EGFP, or pBP-MIR2/EGFP. From each set of transductants, B7.1 molecules were immunoprecipitated and analyzed by Western blot using an anti-Ub antibody.

Mentions: These results demonstrate a striking correlation between the presence of cytosolic lysines, ubiquitination of the cytosolic tail, and the ability to be endocytosed. They reveal that such lysines are necessary for MIR-induced endocytosis but do not establish that they are sufficient for this activity. To examine this question, we created by mutation a stretch of three contiguous lysine residues in the cytoplasmic tail of B7.1, which normally has no lysines in its intracytoplasmic domain and is not endocytosed in response to MIR2 expression (Fig. 5 A). When expressed in cells bearing functional MIR2, the wt molecule is present at readily detectable levels on the cell surface, but the mutant displays clear downregulation (Fig. 5 B). As expected, when the B7.1 KKK mutant was immunoprecipitated with anti-B7.1 and blotted with anti-Ub, ubiquitinated forms of the protein were readily detected, whereas no ubiquitination of wt B7.1 was observed (Fig. 5 C).


A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition.

Coscoy L, Sanchez DJ, Ganem D - J. Cell Biol. (2001)

Addition of lysine into B7.1 enable its downregulation. (A) Sequence of the TM and intracytoplasmic regions of B7.1 (top line) and B7.1KKK mutant (bottom line). The mutant residues within the cytoplasmic region of B7.1KKK are indicated in boldface. (B) B7.1 wt and KKK mutant constructs were stably expressed in HeLa cells. Cells were transduced a second time by pBP-MIR2/EGFP, and surface expression of the B7.1 molecules was analyzed by flow cytometry. (C) Cells expressing B7.1 wt or KKK mutant proteins were stably transduced by pMX-pie, pBP-MIR1/EGFP, or pBP-MIR2/EGFP. From each set of transductants, B7.1 molecules were immunoprecipitated and analyzed by Western blot using an anti-Ub antibody.
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Related In: Results  -  Collection

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fig5: Addition of lysine into B7.1 enable its downregulation. (A) Sequence of the TM and intracytoplasmic regions of B7.1 (top line) and B7.1KKK mutant (bottom line). The mutant residues within the cytoplasmic region of B7.1KKK are indicated in boldface. (B) B7.1 wt and KKK mutant constructs were stably expressed in HeLa cells. Cells were transduced a second time by pBP-MIR2/EGFP, and surface expression of the B7.1 molecules was analyzed by flow cytometry. (C) Cells expressing B7.1 wt or KKK mutant proteins were stably transduced by pMX-pie, pBP-MIR1/EGFP, or pBP-MIR2/EGFP. From each set of transductants, B7.1 molecules were immunoprecipitated and analyzed by Western blot using an anti-Ub antibody.
Mentions: These results demonstrate a striking correlation between the presence of cytosolic lysines, ubiquitination of the cytosolic tail, and the ability to be endocytosed. They reveal that such lysines are necessary for MIR-induced endocytosis but do not establish that they are sufficient for this activity. To examine this question, we created by mutation a stretch of three contiguous lysine residues in the cytoplasmic tail of B7.1, which normally has no lysines in its intracytoplasmic domain and is not endocytosed in response to MIR2 expression (Fig. 5 A). When expressed in cells bearing functional MIR2, the wt molecule is present at readily detectable levels on the cell surface, but the mutant displays clear downregulation (Fig. 5 B). As expected, when the B7.1 KKK mutant was immunoprecipitated with anti-B7.1 and blotted with anti-Ub, ubiquitinated forms of the protein were readily detected, whereas no ubiquitination of wt B7.1 was observed (Fig. 5 C).

Bottom Line: Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells.This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins.Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Microbiology, University of California Medical Center, San Francisco, CA 94143, USA.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells. This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins. Here, we show that expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface. MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity. In vitro, addition of a MIR2-glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion; this reaction is abolished by mutation of the Zn-coordinating residues of the PHD domain. Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

Show MeSH