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Vesicular Proteomics: Isoforms of the COPI and COPII coats - Implications in Protein Sorting and Disease

Rhiel, Manuel

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As for many proteins, subunits of the COPI and COPII vesicular coats have diverged during evolution. To date, multiple isoforms of almost all proteins involved in the formation of these vesicles can be found in higher organisms. While work of others and our own lab has established an important role of the four Sec24 isoforms in the process of cargo/machinery recruitment into COPII vesicles, it remains largely elusive what purpose the isoforms of the coatomer subunits γ- and ζ-COP as well as of the small GTPase Arf (Arf1-6) serve in COPI formation. To investigate a putative role of these proteins in the recruitment of specific proteins into COPI vesicles, and deepen our current understanding of the roles of COPII coat isoforms, we established a method to purify in vitro reconstituted vesicles from SILAC-labeled cells in order to assess and compare their protein content. By doing so we were able to narrow down a concise set of proteins that represents the proteome of these two classes of early secretory pathway vesicular carriers. In contrast to the COPII system, we found that neither isoforms of γ- or ζ-COP, nor any of the four vesicle-generating isoforms of Arf seem to have any influence on the content composition of COPI vesicles. However, while the isoforms of coatomer seem to be capable of recruiting cargo proteins with virtually identical efficacy, Arf1 could be distinguished as the most potent COPI-forming GTPase isoform. We further investigated disease-related mutations identified in one particular Sec24 isoform, Sec24D. These mutations were recently shown to be the sole cause for the development of a syndromic form of osteogenesis imperfecta, which is better known as brittle bone disease. We could show that one single point mutation within Sec24D, the conversion of serine in position 1015 to phenylalanine, completely abrogates the ability of this protein to bind to the ER-Golgi Qa-SNARE protein Syntaxin5 in direct interaction studies. As a consequence, COPII vesicles formed by this Sec24D variant were largely depleted in not only Syntaxin5 but also its partner ER-Golgi SNAREs GS27 and Bet1. Moreover we studied fibroblast cells derived from a patient that carries the Sec24DS1015F mutation in one allele and a point mutation that causes a premature stop-codon within the second allele of the gene. In such cells, the level of all four ER-Golgi SNAREs and also some intra-Golgi SNARE proteins was markedly reduced. However, the distribution of Syntaxin5 under steady state conditions reflected that of control cells.

Item Type: Dissertation
Supervisor: Wieland, Prof. Dr. Felix
Date of thesis defense: 28 February 2018
Date Deposited: 06 Mar 2018 09:16
Date: 2018
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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