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A highly sensitive assay for monitoring the secretory pathway and ER stress. (1/361)

BACKGROUND: The secretory pathway is a critical index of the capacity of cells to incorporate proteins into cellular membranes and secrete proteins into the extracellular space. Importantly it is disrupted in response to stress to the endoplasmic reticulum that can be induced by a variety of factors, including expression of mutant proteins and physiologic stress. Activation of the ER stress response is critical in the etiology of a number of diseases, such as diabetes and neurodegeneration, as well as cancer. We have developed a highly sensitive assay to monitor processing of proteins through the secretory pathway and endoplasmic reticulum (ER) stress in real-time based on the naturally secreted Gaussia luciferase (Gluc). METHODOLOGY/PRINCIPLE FINDINGS: An expression cassette for Gluc was delivered to cells, and its secretion was monitored by measuring luciferase activity in the conditioned medium. Gluc secretion was decreased down to 90% when these cells were treated with drugs that interfere with the secretory pathway at different steps. Fusing Gluc to a fluorescent protein allowed quantitation and visualization of the secretory pathway in real-time. Expression of this reporter protein did not itself elicit an ER stress response in cells; however, Gluc proved very sensitive at sensing this type of stress, which is associated with a temporary decrease in processing of proteins through the secretory pathway. The Gluc secretion assay was over 20,000-fold more sensitive as compared to the secreted alkaline phosphatase (SEAP), a well established assay for monitoring of protein processing and ER stress in mammalian cells. CONCLUSIONS/SIGNIFICANCE: The Gluc assay provides a fast, quantitative and sensitive technique to monitor the secretory pathway and ER stress and its compatibility with high throughput screening will allow discovery of drugs for treatment of conditions in which the ER stress is generally induced.  (+info)

Proteins of the secretory pathway govern virus productivity during lytic gammaherpesvirus infection. (2/361)

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Secretory outposts for the local processing of membrane cargo in neuronal dendrites. (3/361)

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The prodomain of Toxoplasma gondii GPI-anchored subtilase TgSUB1 mediates its targeting to micronemes. (4/361)

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Accessory subunit Ac45 controls the V-ATPase in the regulated secretory pathway. (5/361)

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Mutation in the DC-SIGN cytoplasmic triacidic cluster motif markedly attenuates receptor activity for phagocytosis and endocytosis of mannose-containing ligands by human myeloid cells. (6/361)

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Role of a pro-sequence in the secretory pathway of prothyrotropin-releasing hormone. (7/361)

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A fluorimetry-based ssYFP secretion assay to monitor vasopressin-induced exocytosis in LLC-PK1 cells expressing aquaporin-2. (8/361)

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