Abstract
Human metastatic lymph node (MLN) 64 is composed of two conserved regions. The amino terminus contains a conserved membrane-spanning MENTAL (MLN64 NH(2)-terminal) domain shared with an unique protein called MENTHO (MLN64 NH(2)-terminal domain homologue) and targets the protein to late endosome. The carboxyl-terminal domain is composed of a cholesterol binding steroidogenic acute regulatory-related lipid transfer domain exposed to the cytoplasm. MENTHO overexpression leads to the accumulation of enlarged endosomes. In this study, we show that MLN64 overexpression also induces the formation of enlarged endosomes, an effect that is probably mediated by the MENTAL domain. Using an in vivo photocholesterol binding assay, we find that the MENTAL domain of MLN64 is a cholesterol binding domain. Moreover, glutathione S-transferase pull-down or co-immunoprecipitation experiments demonstrate that this domain mediates homo- and hetero-interaction of MLN64 and MENTHO. In living cells, the expression of paired yellow fluorescent and cyan fluorescent fusion proteins show MENTHO homo-interaction and its interaction with MLN64. These data indicate that within late-endosomal membranes, MLN64 and MENTHO define discrete cholesterol-containing subdomains. The MENTAL domain might serve to maintain cholesterol at the membrane of late endosomes prior to its shuttle to cytoplasmic acceptor(s).
Highlights
Human metastatic lymph node (MLN) 64 is composed of two conserved regions
We show that MLN64 overexpression induces the formation of enlarged endosomes, an effect that is probably mediated by the MENTAL domain
MLN64 and MENTHO Interaction—The MENTAL domain contains four transmembrane helices spanning the membrane of late endosomes [19]
Summary
The amino terminus contains a conserved membrane-spanning MENTAL (MLN64 NH2-terminal) domain shared with an unique protein called MENTHO (MLN64 NH2-terminal domain homologue) and targets the protein to late endosome. The expression of paired yellow fluorescent and cyan fluorescent fusion proteins show MENTHO homo-interaction and its interaction with MLN64 These data indicate that within late-endosomal membranes, MLN64 and MENTHO define discrete cholesterol-containing subdomains. The amino-terminal half of the protein contains four potential transmembrane regions and targets the protein to the membrane of late endosomes, and the carboxylterminal half includes a sterol binding domain [7, 8]. The region conserved between StAR and MLN64, the StAR-related lipid transfer (START) domain, was shown to be a cholesterol binding domain for both proteins [8]. Others have found that the deletion of the START domain
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