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Transport Through the Secretory Pathway of VSVG Tagged With Green Fluorescent Protein: Role of Tubulovesicular Carriers and Microtubules

Published online by Cambridge University Press:  02 July 2020

John Presley ,
Koret Hirschberg  and
Nelson Cole
John Presley
Affiliation:
Cell Biology and Metabolism Branch, Bldg 18T, NICHD, National Institutes of Health, Bethesda, MD20892.
Koret Hirschberg
Affiliation:
Cell Biology and Metabolism Branch, Bldg 18T, NICHD, National Institutes of Health, Bethesda, MD20892.
Nelson Cole
Affiliation:
Cell Biology and Metabolism Branch, Bldg 18T, NICHD, National Institutes of Health, Bethesda, MD20892.
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Extract

The ts045 mutant of VSV G protein has been used in numerous studies to identify biochemical and morphological properties of membrane transport, due to its reversible misfolding and retention in the ER at 40°C and ability to traffic out of the ER and into the Golgi complex upon temperature reduction to 32oC. The dynamic properties of membrane transport intermediates of the secretory pathway, including their lifetime and fate within cells, have not until now been explored due to the inability to follow transport in single living cells. Here, we attached green fluorescent protein to the cytoplasmic tail of VSV G protein in order to visualize ER-to-Golgi and Golgi-to-plasma membrane transport in living cells. VSVG-GFP expressed in Cos cells accumulated in the ER at 40°C and translocated to the Golgi complex when shifted to 32oC. Translocation of the protein was followed using time-lapse imaging of live cells on a confocal microscope. VSVG-GFP accumulated in tubulovesicular structures scattered throughout the cell upon shift from 40°C to 15°C for three hours.

Type
Cell Biology Applications of Green Fluorescent Protein and Other Vital Labeling Probes
Information
Microscopy and Microanalysis , Volume 3 , Issue S2: Proceedings: Microscopy & Microanalysis '97, Microscopy Society of America 55th Annual Meeting, Microbeam Analysis Society 31st Annual Meeting, Histochemical Society 48th Annual Meeting, Cleveland, Ohio, August 10-14, 1997 , August 1997 , pp. 139 - 140
Copyright
Copyright © Microscopy Society of America 1997

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