title: A Novel Two-Tag System for Monitoring Transport and Cleavage through the Classical Secretory Pathway - Adaptation to HIV Envelope Processing.
abstract: The classical secretory pathway is essential for the transport of a host of proteins to the cell surface and/or extracellular matrix. While the pathway is well-established, many factors still remain to be elucidated. One of the most relevant biological processes that occur during transport involves the cleavage of pro-proteins by enzymes residing in the endoplasmic reticulum/Golgi/TransGolgi Network compartment. Teasing out the requirements involved in the classical secretory pathway and cleavage during transport would shed new light into mis-regulation leading to disease. Current methodologies fail to link transport and cleavage at the single cell level. Here, we describe a cell-based assay that relies on an engineered protein scaffold that can discriminate between transport to the cell surface, in the absence or presence of cleavage. Our novel two-tag system works in a robust and quantitative manner and distinguishes between cleaved and non-cleaved events based on cell surface expression of one or two epitope tags, respectively. Here, we have used the HIV-1 envelope as a substrate, which is cleaved during transport, as proof of principle. Importantly, this assay can be easily coupled to existing siRNA-based screens to identify novel regulators and effectors involved in transport and/or cleavage of cell surface proteins. In addition, unlike other in vivo based assays, the assay described here can also be easily adapted to drug discovery purposes.
authors.affiliation: Department of Biology, San Diego State University, San Diego, California, United States of America.
authors.forename: Zachary D
authors.initials: ZD
authors.lastname: Stolp
authors.forename: Aleksandr
authors.initials: A
authors.lastname: Stotland
authors.forename: Samantha
authors.initials: S
authors.lastname: Diaz
authors.forename: Brett J
authors.initials: BJ
authors.lastname: Hilton
authors.forename: Wesley
authors.initials: W
authors.lastname: Burford
authors.forename: Roland
authors.initials: R
authors.lastname: Wolkowicz
mesh_terms: D002460:Cell Line; D005434:Flow Cytometry; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D019302:HIV Envelope Protein gp160 / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D059748:Proteolysis
chemical_list: D015699:HIV Envelope Protein gp120; D019302:HIV Envelope Protein gp160