Regulated motion of glycoproteins revealed by direct visualization of a single cargo in the endoplasmic reticulum

The Journal of Cell Biology
Hisao NagayaIkuo Wada

Abstract

The quality of cargo proteins in the endoplasmic reticulum (ER) is affected by their motion during folding. To understand how the diffusion of secretory cargo proteins is regulated in the ER, we directly analyze the motion of a single cargo molecule using fluorescence imaging/fluctuation analyses. We find that the addition of two N-glycans onto the cargo dramatically alters their diffusion by transient binding to membrane components that are confined by hyperosmolarity. Via simultaneous observation of a single cargo and ER exit sites (ERESs), we could exclude ERESs as the binding sites. Remarkably, actin cytoskeleton was required for the transient binding. These results provide a molecular basis for hypertonicity-induced immobilization of cargo, which is dependent on glycosylation at multiple sites but not the completion of proper folding. We propose that diffusion of secretory glycoproteins in the ER lumen is controlled from the cytoplasm to reduce the chances of aggregation.

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Citations

Aug 20, 2010·Journal of Cell Science·Nikolai I KiskinTom Carter
May 2, 2014·Glycobiology·Masamichi NagaeYoshiki Yamaguchi
Mar 20, 2015·Biological & Pharmaceutical Bulletin·Koji KimuraIkuo Wada
Oct 23, 2012·Journal of Bioscience and Bioengineering·Tsunehito HigashiSachihiro Matsunaga
Feb 9, 2012·The EMBO Journal·Asvin Kk LakkarajuFrançoise Gisou van der Goot
Nov 7, 2009·Biochimica Et Biophysica Acta·Bradley R Pearse, Daniel N Hebert
Jan 20, 2017·Nature Communications·Andreas AnderluhGerhard J Schütz
Nov 17, 2015·Nature Communications·Naokazu InoueIkuo Wada

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Methods Mentioned

BETA
fluorescence correlation spectroscopy
fluorescence microscopy
glycosylation
FCS
transfection

Software Mentioned

SpotTracker2D
MetaMorph
OriginPro
GraphPad
ImageJ
CANVAS
Align Stacks
Prism

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