Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells.
The folding environment in the endoplasmic reticulum (ER) depends on multiple abundant chaperones that function together to accommodate a range of substrates. The ways in which substrate engagement shapes either specific chaperone dynamics or general ER attributes in vivo remain unknown. In this study, we have evaluated how changes in substrate flux through the ER influence the diffusion of both the lectin chaperone calreticulin and an inert reporter of ER crowdedness. During acute changes in substrate load, the inert probe revealed no changes in ER organization, despite significant changes in calreticulin dynamics. By contrast, inhibition of the lectin chaperone system caused rapid changes in the ER environment that could be reversed over time by easing new substrate burden. Our findings provide insight into the normal organization and dynamics of an ER chaperone and characterize the capacity of the ER to maintain homeostasis during acute changes in chaperone activity and availability.
Snapp
Erik L
EL
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, 18 Library Drive, Building 18, Room 101, Bethesda, MD 20892, USA. esnapp@aecom.yu.edu
Sharma
Ajay
A
Lippincott-Schwartz
Jennifer
J
Hegde
Ramanujan S
RS
D000818:Animals; D037282:Calreticulin / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006706:Homeostasis; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D037282:Calreticulin; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins