Abstract
Late endosomal multivesicular bodies (MVBs) are complicated organelles with various subdomains located at the limiting membrane and the internal vesicles (ILVs). ILVs accumulate tetraspanins such as CD63 and CD82 that might form protein assemblies, including major histocompatibility complex class II (MHC-II) and its chaperone human leukocyte antigen (HLA)-DM. Here, we studied the effect of four late endosomal tetraspanin proteins on MHC-II expression. Silencing CD9, CD63 and CD81 enhanced MHC-II expression whereas silencing CD82 did not. No effect on peptide loading was observed. Using confocal FRET technology, we measured the dynamics of CD63 and CD82 interaction with MHC-II and its chaperone HLA-DM. CD63-CD82 interactions remained unaltered in the two MVB subdomains whereas the interactions between CD63 or CD82 homologous pairs changed. CD63 stably associated with MHC-II, and CD82 with HLA-DM, on both MVB subdomains whereas the CD82-MHC-II and CD63-HLA-DM interactions changed. These data visualize for the first time the protein dynamics of tetraspanin assemblies in MVB subdomains. CD63, unlike CD82, stably interacts with MHC-II at both MVB subdomains and controls MHC-II expression.
Highlights
MHC-II-enriched compartments (MIICs) are multivesicular bodies (MVBs) or multilamellar structures of acidic pH that contain major histocompatibility complex class II (MHC-II) and human leukocyte antigen (HLA)-DM molecules, as well as proteases (Neefjes, 1999)
The chaperone HLA-DM resides in MIIC and facilitates the exchange of CLIP for high-affinity binding peptides generated by resident proteases (Denzin and Cresswell, 1995; Kropshofer et al, 1997)
To define the effect of tetraspanin proteins on MHC-II loading and expression, we silenced these in the MHC-II-expressing cell line MelJuSo using RNA interference
Summary
MHC-II-enriched compartments (MIICs) are multivesicular bodies (MVBs) or multilamellar structures of acidic pH that contain major histocompatibility complex class II (MHC-II) and human leukocyte antigen (HLA)-DM molecules, as well as proteases (Neefjes, 1999). Their architecture is unique: a limiting membrane (LM) surrounds a large set of small vesicles termed intraluminal vesicles (ILVs) that might be generated by the ESCRT machinery (Teis et al, 2009). Whereas MHC-II and HLA-DM are both found on the LM and ILV of MVBs, they interact predominantly in the ILV, as determined by fluorescence resonance energy transfer (FRET) experiments (Zwart et al, 2005)
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