The Journal of Cell Biology, Vol 104, 749-760, Copyright © 1987 by The Rockefeller University Press

ARTICLES

Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system

WE Balch, KR Wagner and DS Keller

Transport of the vesicular stomatitis virus-encoded glycoprotein (G protein) between the endoplasmic reticulum (ER) and the cis Golgi compartment has been reconstituted in a cell-free system. Transfer is measured by the processing of the high mannose (man GlcNAc2) ER form of G protein to the man5GlcNAc5 form by the cis Golgi enzyme alpha- mannosidase I. G protein is rapidly and efficiently transported to the Golgi complex by a process resembling that observed in vivo. G protein is trimmed from the high mannose form to the man5GlcNAc2 form without the appearance of the intermediate man GlcNAc2 oligosaccharide species, as is observed in vivo. G protein is found in a sealed membrane-bound compartment before and after incubation. Processing in vitro is sensitive to detergent, and the Golgi alpha-mannosidase I inhibitor 1- deoxymannorjirimycin. Transport between the ER and Golgi complex in vitro requires the addition of a high speed supernatant (cytosol) of cell homogenates, and requires energy in the form of ATP. Efficient reconstitution of export of protein from the ER requires the preparation of homogenates from mitotic cell populations in which the nuclear envelope, ER, and Golgi compartments have been physiologically disassembled before cell homogenization. These results suggest that the high efficiency of transport observed here may require reassembly of functional organelles in vitro.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

• Choe, S. S., Kirkegaard, K. (2004). Intracellular Topology and Epitope Shielding of Poliovirus 3A Protein. J. Virol. 78: 5973-5982 [Abstract] [Full Text]  
• Farmaki, T, Ponnambalam, S, Prescott, A., Clausen, H, Tang, B., Hong, W, Lucocq, J. (1999). Forward and retrograde trafficking in mitotic animal cells. ER-Golgi transport arrest restricts protein export from the ER into COPII-coated structures. J. Cell Sci. 112: 589-600 [Abstract]  
• Pryde, J. G., Farmaki, T., Lucocq, J. M. (1998). Okadaic Acid Induces Selective Arrest of Protein Transport in the Rough Endoplasmic Reticulum and Prevents Export into COPII-Coated Structures. Mol. Cell. Biol. 18: 1125-1135 [Abstract] [Full Text]  
• Lahtinen, U., Dahllof, B., Saraste, J. (1992). Characterization of a 58 kDa cis-Golgi protein in pancreatic exocrine cells. J. Cell Sci. 103: 321-333 [Abstract]  

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents