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Bulk flow revisited: transport of a soluble protein in the secretory pathway.
Traffic. 2009 Dec; 10(12):1819-30
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16 Nov 2009
Confirmation, New Finding
DOI: 10.3410/f.1168077.630195
Using a rapidly folding, non-glycosylated viral protein, the authors measure the rate and efficiency with which this foreign protein traverses the secretory pathway. Secretion is detected within 15 minutes after synthesis, is faster than endogenous proteins, and is highly efficient. These data argue against...Continue reading
Using a rapidly folding, non-glycosylated viral protein, the authors measure the rate and efficiency with which this foreign protein traverses the secretory pathway. Secretion is detected within 15 minutes after synthesis, is faster than endogenous proteins, and is highly efficient. These data argue against a strict requirement for a receptor-based recognition mechanism for rapid and efficient endoplasmic reticulum (ER) export.
Protein export from the ER utilizes a quality control machinery to ensure that only properly folded proteins are released. Rate-limiting steps for protein secretion are the folding and assembly steps that include disulfide bond formation, glycosylation, and multimeric association. Thor et al. present a novel system that enables them to monitor the progress of a completely foreign protein through the ER and Golgi of Chinese hamster ovary (CHO) and Madin-Darby canine kidney (MDCK) cells. They follow the export of an HA-tagged, signal sequence-bearing, C-terminal domain of the Semliki Forest virus capsid protein that is normally cytoplasmic in origin. The protein is known to fold co-translationally and in a chaperone-independent manner; it is also an auto-protease, and its self-cleavage can be used to report on its folding completion. The protein folds within 1 minute, appears in the medium linearly from 15 minutes to two hours, and 85% is released. It uses the normal secretory pathway because secretion is blocked by Brefeldin-A and requires ATP. Current models for ER export invoke sorting receptors, and there exist several well-established examples of coat-cargo or lectin-cargo interactions that facilitate ER export in specific cases. But it is difficult to imagine receptors for all cargoes, and this study suggests that receptors may not always be required. The absence of a receptor requirement would help explain the ability of eukaryotic cells to secrete foreign proteins, including green fluorescent protein (GFP), bacterial beta-lactamase and, from this elegant work, Semliki Forest virus capsid protein.
Protein export from the ER utilizes a quality control machinery to ensure that only properly folded proteins are released. Rate-limiting steps for protein secretion are the folding and assembly steps that include disulfide bond formation, glycosylation, and multimeric association. Thor et al. present a novel system that enables them to monitor the progress of a completely foreign protein through the ER and Golgi of Chinese hamster ovary (CHO) and Madin-Darby canine kidney (MDCK) cells. They follow the export of an HA-tagged, signal sequence-bearing, C-terminal domain of the Semliki Forest virus capsid protein that is normally cytoplasmic in origin. The protein is known to fold co-translationally and in a chaperone-independent manner; it is also an auto-protease, and its self-cleavage can be used to report on its folding completion. The protein folds within 1 minute, appears in the medium linearly from 15 minutes to two hours, and 85% is released. It uses the normal secretory pathway because secretion is blocked by Brefeldin-A and requires ATP. Current models for ER export invoke sorting receptors, and there exist several well-established examples of coat-cargo or lectin-cargo interactions that facilitate ER export in specific cases. But it is difficult to imagine receptors for all cargoes, and this study suggests that receptors may not always be required. The absence of a receptor requirement would help explain the ability of eukaryotic cells to secrete foreign proteins, including green fluorescent protein (GFP), bacterial beta-lactamase and, from this elegant work, Semliki Forest virus capsid protein.
Disclosures
None declared
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Pfeffer S: F1000Prime Recommendation of [Thor F et al., Traffic 2009, 10(12):1819-30]. In F1000Prime, 16 Nov 2009; DOI: 10.3410/f.1168077.630195. F1000Prime.com/1168077#eval630195
F1000Prime Recommendations, Dissents and Comments for [Thor F et al., Traffic 2009, 10(12):1819-30]. In F1000Prime, 25 May 2013; F1000Prime.com/1168077
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The C-terminal domain, Cp, of the Semliki Forest virus capsid protein, known for its rapid, efficient and chaperone-independent folding, was used to measure bulk fluid flow in the secretory pathway of Chinese hamster ovary cells. Being small, nonglycosylated, soluble and cytoplasmic in origin, Cp was not likely to interact with lectins, cargo receptors and retention factors. Using pulse-chase analysis, we observed that translocation into the endoplasmic reticulum resulted in rapid and efficient folding and transport of the newly synthesized Cp protein to the extracellular medium. The first Cp molecules were secreted 15 min after synthesis, which is the fastest transport of a protein so far recorded in mammalian cells. The rate constant of secretion was 1.2% per min, which amounts to an estimated bulk flow rate of about 155 coat protein II (COPII) vesicles per second. Transport was independent of expression level, and blocked by CI-976, brefeldin A and ATP depletion indicating that it depended on COPII vesicle formation, and followed the classical secretory pathway. In polarized Madin-Darby canine kidney cells, the secretion rate was similar but occurred mainly apically. The results demonstrated that fluid flow in the secretory pathway is fast, and can therefore play a significant role in the secretion of soluble secretory products.
DOI: 10.1111/j.1600-0854.2009.00989.x
PMID: 19843282
