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

Ishido Miwako; Sandra L. Schmid

doi:10.1016/j.yexcr.2005.12.022

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

Ishido Miwako, Sandra L. Schmid

Research output: Contribution to journal › Article › peer-review

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 language	English (US)
Pages (from-to)	1335-1344
Number of pages	10
Journal	Experimental Cell Research
Volume	312
Issue number	8
DOIs	https://doi.org/10.1016/j.yexcr.2005.12.022
State	Published - 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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10.1016/j.yexcr.2005.12.022

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

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.",

keywords = "AP2, Cell-free assay, Clathrin, Dynamin, Endocytosis, LDL-receptor related protein",

author = "Ishido Miwako and Schmid, {Sandra L.}",

note = "Funding Information: We thank Dr. Sean Conner for providing purified CRMP-2 and Schmid laboratory members for critically reading the manuscript. We also thank Leslie Nielsen, The Department of Animal Resources and Dr. Sunia Trauger, Scripps Center for Mass Spectrometry for their help. The research was supported by NIH Grant R37MH61345 to SLS. I. Miwako was supported by an American Heart Association Postdoctoral Fellowship No. 0325053Y. This is TSRI manuscript number 17561-CB.",

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AU - Miwako, Ishido

AU - Schmid, Sandra L.

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PY - 2006/5/1

Y1 - 2006/5/1

N2 - 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.

AB - 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.

KW - AP2

KW - Cell-free assay

KW - Clathrin

KW - Dynamin

KW - Endocytosis

KW - LDL-receptor related protein

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

Abstract

Keywords

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