Functional Characterization of a Lysosomal Sorting Motif in the Cytoplasmic Tail of HLA-DOβ

Alexandre Brunet,Angela Samaan,Francis Deshaies,Thomas J Kindt,Jacques Thibodeau

doi:10.1074/jbc.m005112200

Abstract

HLA-DO is an intracellular non-classical class II major histocompatibility complex molecule expressed in the endocytic pathway of B lymphocytes, which regulates the loading of antigenic peptides onto classical class II molecules such as HLA-DR. The activity of HLA-DO is mediated through its interaction with the peptide editor HLA-DM. Here, our results demonstrate that although HLA-DO is absolutely dependent on its association with DM to egress the endoplasmic reticulum, the cytoplasmic portion of its beta chain encodes a functional lysosomal sorting signal. By confocal microscopy and flow cytometry analysis, we show that reporter transmembrane molecules fused to the cytoplasmic tail of HLA-DObeta accumulated in Lamp-1(+) vesicles of transfected HeLa cells. Mutagenesis of a leucine-leucine motif abrogated lysosomal accumulation and resulted in cell surface redistribution of reporter molecules. Finally, we show that mutation of the di-leucine sequence in DObeta did not alter its lysosomal sorting when associated with DM molecules. Taken together, these results demonstrate that lysosomal expression of the DO-DM complex is mediated primarily by the tyrosine-based motif of HLA-DM and suggest that the DObeta-encoded motif is involved in the fine-tuning of the intracellular sorting.

Highlights

Introduction of mutations into theDO␤ cytoplasmic tail cDNA sequence was performed by PCR overlap extension [50]
By confocal microscopy and flow cytometry analysis, we show that reporter transmembrane molecules fused to the cytoplasmic tail of HLA-DO␤ accumulated in Lamp-1؉ vesicles of transfected HeLa cells
As wild-type DR␣/DR␤ (DR1) molecules did not accumulate in intracellular compartments under the same experimental conditions, we concluded that a lysosomal sorting motif was encoded in the HLA-DO␤ chain

Summary

Introduction

Introduction of mutations into theDO␤ cytoplasmic tail cDNA sequence was performed by PCR overlap extension [50]. The 3Ј PCR product was generated using the complementary mutagenic primers (DO␤Y227ABspEIc, 5ЈCAG AAA GGA GCT GTC CGG ACG-3Ј; DO␤T230LPstIc, 5Ј-TAT GTG AGG CTG CAG ATG TCT G-3Ј; and DO␤LL242–243AAXbaIc, 5Ј-GTC TCT AGA GCT GTT GCG GCC CCT CAG TCA-3Ј) and DR␤008 3Ј (5Ј-ACT CGA TCT TTG AGA AAC ATT-3Ј) which hybridized to the non-coding 3Ј end of the cDNA. The two overlapping PCR products were mixed, and a final PCR was performed using the flanking primers. This product was subsequently subcloned into SacI and HindIII sites of RSV.. DR18/ DO␤LL242–243AA) by replacing the EcoRV-HindIII fragment with the equivalent fragment from mutated DO␤ cDNA in RSV. described above. Underlined nucleotides in primer sequences correspond to mutations introduced in the PCR products

Methods

Results

Conclusion