Contribution of the clathrin adaptor AP-1 subunit µ1 to acidic cluster protein sorting

J Cell Biol. 2017 Sep 4;216(9):2927-2943. doi: 10.1083/jcb.201602058. Epub 2017 Jul 25.

Abstract

Acidic clusters act as sorting signals for packaging cargo into clathrin-coated vesicles (CCVs), and also facilitate down-regulation of MHC-I by HIV-1 Nef. To find acidic cluster sorting machinery, we performed a gene-trap screen and identified the medium subunit (µ1) of the clathrin adaptor AP-1 as a top hit. In µ1 knockout cells, intracellular CCVs still form, but acidic cluster proteins are depleted, although several other CCV components were either unaffected or increased, indicating that cells can compensate for long-term loss of AP-1. In vitro experiments showed that the basic patch on µ1 that interacts with the Nef acidic cluster also contributes to the binding of endogenous acidic cluster proteins. Surprisingly, µ1 mutant proteins lacking the basic patch and/or the tyrosine-based motif binding pocket could rescue the µ1 knockout phenotype completely. In contrast, these mutants failed to rescue Nef-induced down-regulation of MHC class I, suggesting a possible mechanism for attacking the virus while sparing the host cell.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Protein Complex 1 / chemistry
  • Adaptor Protein Complex 1 / genetics
  • Adaptor Protein Complex 1 / metabolism*
  • Adaptor Protein Complex mu Subunits / chemistry
  • Adaptor Protein Complex mu Subunits / genetics
  • Adaptor Protein Complex mu Subunits / metabolism*
  • CRISPR-Cas Systems
  • Clathrin-Coated Vesicles / metabolism*
  • Flow Cytometry
  • Gene Knockdown Techniques
  • Genotype
  • HEK293 Cells
  • HIV-1 / genetics
  • HIV-1 / metabolism*
  • HeLa Cells
  • Histocompatibility Antigens Class I / genetics
  • Histocompatibility Antigens Class I / metabolism
  • Host-Pathogen Interactions
  • Humans
  • Models, Molecular
  • Mutation
  • Phenotype
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Transport
  • Structure-Activity Relationship
  • Time Factors
  • Transfection
  • nef Gene Products, Human Immunodeficiency Virus / chemistry
  • nef Gene Products, Human Immunodeficiency Virus / genetics
  • nef Gene Products, Human Immunodeficiency Virus / metabolism*

Substances

  • AP1M1 protein, human
  • Adaptor Protein Complex 1
  • Adaptor Protein Complex mu Subunits
  • Histocompatibility Antigens Class I
  • nef Gene Products, Human Immunodeficiency Virus
  • nef protein, Human immunodeficiency virus 1