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Updated: Aug 13, 2026

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays
Published on: September 28, 2018
A cell-free biochemical complementation assay reveals complex and redundant cytosolic requirements for LRP
Ishido Miwako1, Sandra L Schmid
1Department of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.
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.
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