A cell-free biochemical complementation assay reveals complex and redundant cytosolic requirements for LRP endocytosis

Ishido Miwako, Sandra L. Schmid

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

The low density lipoprotein receptor-related protein (LRP) binds multiple, distinct ligands and participates in constitutive endocytosis and signal transduction. Using an in vitro reconstitution system and a new biochemical complementation assay, we have explored the limiting cytosolic requirements for endocytosis of LRP from isolated plasma membranes. We find that clathrin, AP2 and dynamin do not support efficient LRP uptake and that additional factors present in a 30% ammonium sulfate supernatant fraction of bovine brain cytosol (AS supt) are required. Fractionation of the AS supt revealed that multiple and redundant factors are required to support LRP endocytosis. Among these, we identified Hsc70, synaptojanin1 and CRMP-2 by mass spectrometry. Our data suggest that LRP, which bears several distinct endocytic motifs in its cytoplasmic domain, may use multiple pathways for endocytosis in vitro.

Original languageEnglish (US)
Pages (from-to)1335-1344
Number of pages10
JournalExperimental Cell Research
Volume312
Issue number8
DOIs
StatePublished - May 1 2006

Keywords

  • AP2
  • Cell-free assay
  • Clathrin
  • Dynamin
  • Endocytosis
  • LDL-receptor related protein

ASJC Scopus subject areas

  • Cell Biology

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