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.
Highlights
The classical secretory pathway is critical for normal cell function as it is utilized for the transport of many proteins to the cell membrane or for their secretion into the extracellular matrix (ECM)
Our assay relies on a double-tagged scaffold that is used to discriminate between cleaved and non-cleaved events that occur in the Endoplasmic Reticulum (ER)/Golgi/Trans-Golgi Network (TGN) compartment during transport to the cell surface
We have developed a novel in vivo cell-based assay that discriminates between cleaved and non-cleaved events that occur in the ER/Golgi/TGN apparatus
Summary
The classical secretory pathway is critical for normal cell function as it is utilized for the transport of many proteins to the cell membrane or for their secretion into the extracellular matrix (ECM). In order to reach the secretory pathway, proteins are targeted to the Endoplasmic Reticulum (ER), and travel through the Golgi and Trans-Golgi Network (TGN) where they can be co- and post-translationally modified into mature forms prior to their final destination; insertion within cellular membranes or secretion [1]. Proteolytic processing within the secretory pathway relies on a wide array of ER/Golgi/TGN resident cellular proteases, including signal peptidases (SP), signal peptide peptidases (SPP), and proprotein convertases (PC) [5,6,7]. ER/Golgi/TGN resident proteases play an essential role in normal function, and mis-regulation of these enzymes has been attributed to the development of cancer, Alzheimer’s, and other diseases [8,9]
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