Khalil Hayssam, Brunet Alexandre, Thibodeau Jacques
Laboratoire d'Immunologie Moléculaire, Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, Canada, H3C 3J7.
J Cell Sci. 2005 Oct 15;118(Pt 20):4679-87. doi: 10.1242/jcs.02592. Epub 2005 Sep 27.
The p35 isoform of the human invariant chain (Iip35) contains an N-terminal RXR endoplasmic-reticulum (ER) retention signal that becomes nonfunctional only after assembly with MHC-class-II molecules. We have previously shown that the MHC-class-II beta-chain cytoplasmic tail is crucial for the maturation of class-II/Iip35 complexes. In order to shed some light on the molecular determinants involved in shielding the RXR motif, we performed site-directed mutagenesis of the DRbeta chain and Ii cytoplasmic domains. Chimeric beta chains with irrelevant cytoplasmic tails allowed the efficient transport of Iip35 out of the ER in transiently transfected HEK 293T cells. An alanine scan of the cytoplasmic tail of HLA-DRbeta confirmed that no specific motif is required to overcome ER retention. Surprisingly, a beta chain with a three-amino-acid-long cytoplasmic tail (Tyr-Phe-Arg) was sufficient to overcome the Iip35 RXR motif. Moreover, replacement of residues F231 and R232 with alanines created a cytoplasmic tail (Tyr-Ala-Ala) that allowed ER egress. Given the limited length of this tail, steric hindrance would only be possible if the Ii ER retention motif was close to the membrane in the first place. However, this is not likely because an Ii molecule with an internal cytoplasmic deletion bringing the RXR motif closer to the membrane is not retained in the ER, even in the absence of class-II molecules. These results suggest that MHC-class-II molecules overcome ER retention and prevent COPI binding to the Iip35 RXR motif through a mechanism distinct from steric hindrance by its beta chain.
人类恒定链的p35亚型（Ii p35）含有一个N端RXR内质网（ER）滞留信号，该信号只有在与MHC-II类分子组装后才会失去功能。我们之前已经表明，MHC-II类β链细胞质尾巴对于II类/Ii p35复合物的成熟至关重要。为了阐明参与屏蔽RXR基序的分子决定因素，我们对DRβ链和Ii细胞质结构域进行了定点诱变。带有不相关细胞质尾巴的嵌合β链能够使Ii p35在瞬时转染的HEK 293T细胞中有效地从内质网转运出来。对HLA-DRβ细胞质尾巴的丙氨酸扫描证实，不需要特定基序来克服内质网滞留。令人惊讶的是，具有三个氨基酸长细胞质尾巴（Tyr-Phe-Arg）的β链足以克服Ii p35的RXR基序。此外，将F231和R232残基替换为丙氨酸产生了一个允许内质网出芽的细胞质尾巴（Tyr-Ala-Ala）。鉴于这个尾巴的长度有限，只有当Ii内质网滞留基序首先靠近膜时，空间位阻才有可能。然而，这不太可能，因为即使在没有II类分子的情况下，具有内部细胞质缺失从而使RXR基序更靠近膜的Ii分子也不会保留在内质网中。这些结果表明，MHC-II类分子通过一种不同于其β链空间位阻的机制克服内质网滞留并阻止COPI与Ii p35的RXR基序结合。
