The development of cell-free systems to elucidate the components involved in the endomembrane trafficking of viral proteins between the endoplasmic reticulum and Golgi apparatus

Mark Andrew Paulik, Purdue University

Abstract

The objectives of the study were to elucidate some of the requirements and components involved in the budding, intracellular movement and specificity of membrane fusion involved in endomembrane trafficking of viral glycoproteins between the transitional endopolasmic reticulum (TER) and Golgi apparatus (GA) in a cell-free system. A cell-free vesicle budding assay was developed in order to elucidate the requirements for vesicle release from the TER. Transition vesicle formation was temperature- and nucleoside triphosphate-dependent and required a cytosolic fraction. A cell-free transfer system consisting of the transfer of vesicular stomatitis virus G protein (VSV-G) from the TER of VSV-G infected BHK cells to cis GA of rat liver immobilized on nitrocellulose was developed. The VSV-G cell-free transfer showed similar temperature sensitivity, donor specificity, requirement for ATP and response to inhibitors to that exhibited by transfer and processing of VSV-G protein in vivo. Another cell-free transfer system which utilized an ELISA assay for detection of feline immunodeficiency virus (FIV) envelope glycoprotein transfer was developed to measure vesicular transfer between the ER and cis GA. The transfer of the FIV envelope glycoprotein was ATP- and cytosol-dependent. The transfer was specific for cis GA. Since transfer was specific for cis GA in the cell-free systems described, evidence was sought for a cis GA-specific "docking" protein capable of specifically binding transition vesicles. p38cis was an extrinsic membrane protein which was localized in membranes of cis GA and was not found in trans GA cisternae when analyzed by SDS-PAGE. Peptide sequence was obtained and antisera to one of the peptides, when added to the cell-free transfer system, inhibited the transfer of membrane from TER to cis GA. The antisera prevented not the formation of transition vesicles, but their subsequent fusion with the cis GA. Peptide-specific antisera directed against the consensus GTP-binding region (GAGES) of heterotrimeric G$\alpha$ cross reacted with p38cis suggesting that p38cis also is a GTP-binding protein. Thus, p38cis may be a candidate for the docking protein responsible for the correct targeting of transition vesicles to cis GA.

Degree

Ph.D.

Advisors

Morre, Purdue University.

Subject Area

Cellular biology|Biochemistry

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