Recombinant heptameric coatomer complexes: novel tools to study isoform-specific functions.
Traffic. 2011 Jun; 12(6):682-92
This paper describes an important technical advance with wide-ranging implications for those interested in the structure and function of the coat protein I (COPI) vesicle coat complex -- coatomer. The paper is significant both for its technical accomplishment and the expression and purification of recombinant coatomer from insect cells (which comprises seven proteins and weighs in at more than 500kDa) but also because it provides a means by which to explore the roles of isoforms of mammalian coatomer. In addition, extending such an approach to coatomer from other organisms, such as yeast, will allow purification of mutant forms of the complex, which might otherwise not be feasible.
Banfield D: F1000Prime Recommendation of [Sahlmüller MC et al., Traffic 2011, 12(6):682-92]. In F1000Prime, 26 Aug 2011; DOI: 10.3410/f.12992956.14301054. F1000Prime.com/12992956#eval14301054
F1000Prime Recommendations, Dissents and Comments for [Sahlmüller MC et al., Traffic 2011, 12(6):682-92]. In F1000Prime, 08 Dec 2013; F1000Prime.com/12992956
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COPI (coat protein I)-coated vesicles are implicated in various transport steps within the early secretory pathway. The major structural component of the COPI coat is the heptameric complex coatomer (CM). Recently, four isoforms of CM were discovered that may help explain various transport steps in which the complex has been reported to be involved. Biochemical studies of COPI vesicles currently use CM purified from animal tissue or cultured cells, a mixture of the isoforms, impeding functional and structural studies of individual complexes. Here we report the cloning into single baculoviruses of all CM subunits including their isoforms and their combination for expression of heptameric CM isoforms in insect cells. We show that all four isoforms of recombinant CM are fully functional in an in vitro COPI vesicle biogenesis assay. These novel tools enable functional and structural studies on CM isoforms and their subcomplexes and allow studying mutants of CM.
© 2011 John Wiley & Sons A/S.
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