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PMID: 9490717 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

A dominant-negative clathrin mutant differentially affects trafficking of molecules with distinct sorting motifs in the class II major histocompatibility complex (MHC) pathway.

The Journal of cell biology ·Vol. 140 ·No. 5 ·1998-03-09 ·Pages 1023-37

Liu SH, Marks MS, Brodsky FM

Abstract

The role of clathrin in intracellular sorting was investigated by expression of a dominant-negative mutant form of clathrin, termed the hub fragment. Hub inhibition of clathrin-mediated membrane transport was established by demonstrating a block of transferrin internalization and an alteration in the intracellular distribution of the cation-independent mannose-6-phosphate receptor. Hubs had no effect on uptake of FITC-dextran, adaptor distribution, organelle integrity in the secretory pathway, or cell surface expression of constitutively secreted molecules. Hub expression blocked lysosomal delivery of chimeric molecules containing either the tyrosine-based sorting signal of H2M or the dileucine-based sorting signal of CD3gamma, confirming a role for clathrin-coated vesicles (CCVs) in recognizing these signals and sorting them to the endocytic pathway. Hub expression was then used to probe the role of CCVs in targeting native molecules bearing these sorting signals in the context of HLA-DM and the invariant chain (I chain) complexed to HLA-DR. The distribution of these molecules was differentially affected. Accumulation of hubs before expression of the DM dimer blocked DM export from the TGN, whereas hubs had no effect on direct targeting of the DR-I chain complex from the TGN to the endocytic pathway. However, concurrent expression of hubs, such that hubs were building to inhibitory concentrations during DM or DR-I chain expression, caused cell surface accumulation of both complexes. These observations suggest that both DM and DR-I chain are directly transported to the endocytic pathway from the TGN, DM in CCVs, and DR-I chain independent of CCVs. Subsequently, both complexes can appear at the cell surface from where they are both internalized by CCVs. Differential packaging in CCVs in the TGN, mediated by tyrosine- and dileucine-based sorting signals, could be a mechanism for functional segregation of DM from DR-I chain until their intended rendezvous in late endocytic compartments.

MeSH Terms
Adaptor Protein Complex alpha Subunits Adaptor Proteins, Vesicular Transport Animals Binding Sites Biological Transport Cattle Clathrin/biosynthesis,genetics,metabolism Clathrin Heavy Chains Coated Vesicles/metabolism Endocytosis/physiology Gene Expression HLA-D Antigens/metabolism HLA-DR Antigens/metabolism HeLa Cells Histocompatibility Antigens Class II Humans Intracellular Fluid/metabolism Leucine/metabolism Membrane Proteins/metabolism Mutagenesis Recombinant Fusion Proteins/metabolism Transfection Tyrosine/metabolism
Chemicals
Adaptor Protein Complex alpha Subunits Adaptor Proteins, Vesicular Transport Clathrin H2-M antigens HLA-D Antigens HLA-DM antigens HLA-DR Antigens Histocompatibility Antigens Class II Membrane Proteins Recombinant Fusion Proteins Clathrin Heavy Chains Tyrosine Leucine
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Liu S H
The G.W. Hooper Foundation, Department of Microbiology and Immunology, University of California, San Francisco, California 94143-0552, USA.
Marks M S
Brodsky F M
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.140.5.1023
Published
1998-03-09
Pages
1023-37
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2132696
Subset
IM
Grants
NIGMS NIH HHS · GM57657 · United States
NIGMS NIH HHS · GM38093 · United States
NIAID NIH HHS · AI39152 · United States
External Links
PubMed source DOI: 10.1083/jcb.140.5.1023 PMC Full Text View journal details
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