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© The Rockefeller University Press, 0021-9525/1998//1023 $5.00
The Journal of Cell Biology, Volume 140, Number 5, , 1998 1023-1037

Article

A Dominant-negative Clathrin Mutant Differentially Affects Trafficking of Molecules with Distinct Sorting Motifs in the Class II Major Histocompatibility Complex (MHC) Pathway



Shu-Hui Liu*, Michael S. Marks{ddagger}, and Frances M. Brodsky*

* The G.W. Hooper Foundation, Department of Microbiology and Immunology, and Department of Biopharmaceutical Sciences, and Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0552; and {ddagger} Department of Pathology and Laboratory Medicine, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104-6082

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 CD3{gamma}, 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.

Abbreviations used in this paper: AP, adaptor protein; CCV, clathrin-coated vesicle; CI-M6PR, cation-independent mannose-6-phosphate receptor; CMV, cytomegalovirus; COP, coat protein; FITC-Tfn, fluorescein-conjugated transferrin; I chain, invariant chain; IL, interleukin; LC, light chain; LRSC, lissamine rhodamine; PM, plasma membrane; TfnR, transferrin; TfnR, transferrin receptor.

Address all correspondence to Frances M. Brodsky, The G.W. Hooper Foundation, Department of Microbiology and Immunology and Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0552. Tel.: (415) 476-6406. Fax: (415) 476-6185. E-mail: fmarbro{at}itsa.ucsf.edu


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