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Novel Insights into the Mechanisms Involved in the Biogenesis of Copii-Coated Vesicles

Title: Novel Insights into the Mechanisms Involved in the Biogenesis of Copii-Coated Vesicles.
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Name(s): Hariri, Hanaa H., author
Stagg, Scott, professor directing dissertation
Schlenoff, Joseph B., university representative
Miller, Brian G., committee member
Stroupe, Margaret Elizabeth, committee member
Keller, Thomas C. S., committee member
Florida State University, degree granting institution
College of Arts and Sciences, degree granting college
Program in Molecular Biophysics, degree granting department
Type of Resource: text
Genre: Text
Issuance: monographic
Date Issued: 2014
Publisher: Florida State University
Place of Publication: Tallahassee, Florida
Physical Form: computer
online resource
Extent: 1 online resource (138 pages)
Language(s): English
Abstract/Description: The trafficking of proteins between intracellular compartments must be tightly regulated in order to maintain cellular balance. Early secretion form the endoplasmic reticulum is mediated by a complex of five coat proteins forming the COPII coat. Genetic, biochemical, and structural studies have resulted in a model for the formation of COPII-coated vesicles. Early stages of COPII assembly involve activation of small guanosine triphosphatase protein Sar1 which cycles between two conformations based on its nucleotide state. Upon exchanging GDP for GTP, Sar1 exposes an amphipathic N-terminal α-helix which inserts into the outer leaflet of the ER membrane initiating curvature. This step is followed by the sequential assembly of Sec23/24 and Sec13/31 protein complexes forming a coat on the ER membrane that recruits and packages the cargo to be transported. Nearly one third of the protein encoded by the mammalian genome are trafficked from the ER through the secretory pathway. Mutations in the COPII proteins lead to multiple diseases that are associated with the retention of cargo proteins at their site of synthesis in the endoplasmic reticulum. Thus, the COPII machinery must adapted to transport highly heterogeneous sets of proteins in terms of their shapes, sizes, topology and final destinations. However, the mechanism by which this occurs remains largely unknown. Answering this question requires a deep understanding not only of the roles played by different COPII components in modulating lipid membrane curvature and in inducing vesicle scission, but also the interactions between cargo proteins and the COPII coat components. In this study I first addressed the role of Sar1 and GTP hydrolysis in membrane deformation and vesicle scission. Sar1 alone is found competent for vesicle scission in a manner that depends on the concentration of Sar1 molecules occupying the membrane. The mechanism by which this occurs involves the alignment of Sar1 molecules on low curvature membranes to form an extended lattice. The continuity of this lattice breaks down as the curvature locally increases. This correlation between Sar1 lattice formation and the degree of curvature implicates Sar1 organization in regulating membrane constriction prior to vesicle scission. The smallest repeating unit constituting the ordered lattice is a Sar1 dimer. Therefore, similar to other membrane shaping proteins, Sar1 dimerization may be responsible for the formation of constrictive membrane curvature. Based on this, initial predictions suggested that directed mutagenesis of the sites of Sar1 dimerization will generate Sar1 mutants that are incapable of performing vesicle scission. We propose a model whereby Sar1 dimers assemble into ordered arrays to promote membrane constriction and COPII-directed vesicle scission. In the absence of membranes, valuable insights into the interactions of the COPII proteins in an assembled coat are derived from structural analysis using cryogenic electron microscopy. Therefore, understanding the requirements for COPII vesicle scission in vitro is also critical for generating a specimen of COPII-coated vesicles budded from ER-derived microsomes and suitable for further structural analysis.
Identifier: FSU_migr_etd-9184 (IID)
Submitted Note: A Dissertation submitted to the Institute of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Degree Awarded: Fall Semester, 2014.
Date of Defense: November 3, 2014.
Keywords: COPII, Mechanism, Protein, Scission, Secretion, Vesicles
Bibliography Note: Includes bibliographical references.
Advisory Committee: Scott Stagg, Professor Directing Dissertation; Brian Miller, Committee Member; Beth Stroupe, Committee Member; Thomas Keller, Committee Member.
Subject(s): Biophysics
Biochemistry
Biology
Persistent Link to This Record: http://purl.flvc.org/fsu/fd/FSU_migr_etd-9184
Owner Institution: FSU

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Hariri, H. H. (2014). Novel Insights into the Mechanisms Involved in the Biogenesis of Copii-Coated Vesicles. Retrieved from http://purl.flvc.org/fsu/fd/FSU_migr_etd-9184