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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2016)

The coat protein complex II (COPII) achieves cargo sorting and vesicle formation at the endoplasmic reticulum (ER) and aids in protein trafficking by the early secretory pathway. The flexibility of the coat machinery to accommodate diverse size and shaped cargo in order to meet the demands of the cell is not well understood. An example of a large cargo protein is collagen, which is synthesized as 300nm precursor fibrils of procollagen in the endoplasmic reticulum (ER). The molecular mechanism by which large procollagen fibrils are accommodated in canonical COPII vesicles of about 70nm remains unclear. Genetic and biochemical evidence for the role of COPII in collagen trafficking has been established in several studies. Recent collaborative work has shown that mono-ubiquitylation of Sec31 by the ubiquitin ligase complex Cul3-Klhl12 leads to the formation of large COPII vesicles (200-500nm) that drive the secretion of collagen. Amongst the follow-up questions from this study were, whether these enlarged vesicles are bona fide transport carriers of procollagen and what might the three-dimensional ultrastructure be. This dissertation work aims to characterize the role of large COPII coated vesicles in intracellular procollagen-1 trafficking. Using a combination of in vivo imaging techniques and in vitro cell-free reconstitution assays, I present evidence that addresses this question.

    Cover page: The role of COPII coated vesicles in procollagen-1 trafficking
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2019)
    COPII-coated vesicles are the primary mediators of ER-to-Golgi trafficking. Sar1, one of the five core COPII components, is a highly conserved small GTPase, which, upon GTP binding, recruits the other COPII proteins to the ER membrane. It has been hypothesized that the changes in the kinetics of SAR1 GTPase may allow for the secretion of large cargoes. Here we developed a cell-free assay to recapitulate COPII-dependent budding of large lipoprotein cargoes from the ER. We identified fatty-acid binding protein 5 (FABP5) as an enhancer of this budding process. We found that FABP5 promotes the budding of particles ∼150 nm in diameter and modulates the kinetics of the SAR1 GTPase cycle. We further found that FABP5 enhances the trafficking of lipoproteins and of other cargoes, including collagen. These data identify a novel regulator of SAR1 GTPase activity and highlight the importance of this activity for trafficking of large cargoes.
      Cover page: Fatty-acid binding protein 5 modulates the SAR1 GTPase cycle and enhances budding of large COPII cargoes
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2017)
      The coat protein complex II (COPII) is essential for the transport of large cargo, such as 300-nm procollagen I (PC1) molecules, from the endoplasmic reticulum (ER) to the Golgi. Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles at ER exit sites to more than 300 nm in diameter and accelerates the secretion of PC1. However, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy, correlated light electron microscopy, and live-cell imaging, we demonstrate the existence of mobile COPII-coated vesicles that completely encapsulate the cargo PC1 and are physically separated from ER. We also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude that large COPII vesicles are bona fide carriers of PC1.
        Cover page: COPII-coated membranes function as transport carriers of intracellular procollagen I
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2012)
        Packaging of proteins from the endoplasmic reticulum into COPII vesicles is essential for secretion. In cells, most COPII vesicles are approximately 60-80 nm in diameter, yet some must increase their size to accommodate 300-400 nm procollagen fibres or chylomicrons. Impaired COPII function results in collagen deposition defects, cranio-lenticulo-sutural dysplasia, or chylomicron retention disease, but mechanisms to enlarge COPII coats have remained elusive. Here, we identified the ubiquitin ligase CUL3-KLHL12 as a regulator of COPII coat formation. CUL3-KLHL12 catalyses the monoubiquitylation of the COPII-component SEC31 and drives the assembly of large COPII coats. As a result, ubiquitylation by CUL3-KLHL12 is essential for collagen export, yet less important for the transport of small cargo. We conclude that monoubiquitylation controls the size and function of a vesicle coat.
          Cover page: Ubiquitin-dependent regulation of COPII coat size and function.
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2014)
          Myxococcus xanthus is a bacterial micro-predator known for hunting other microbes in a wolf pack-like manner. Outer membrane vesicles (OMVs) are produced in large quantities by M. xanthus and have a highly organized structure in the extracellular milieu, sometimes occurring in chains that link neighboring cells within a biofilm. OMVs may be a vehicle for mediating wolf pack activity by delivering hydrolytic enzymes and antibiotics aimed at killing prey microbes. Here, both the protein and small molecule cargo of the OMV and membrane fractions of M. xanthus were characterized and compared. Our analysis indicates a number of proteins that are OMV-specific or OMV-enriched, including several with putative hydrolytic function. Secondary metabolite profiling of OMVs identifies 16 molecules, many associated with antibiotic activities. Several hydrolytic enzyme homologs were identified, including the protein encoded by MXAN_3564 (mepA), an M36 protease homolog. Genetic disruption of mepA leads to a significant reduction in extracellular protease activity suggesting MepA is part of the long-predicted (yet to date undetermined) extracellular protease suite of M. xanthus.
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2014)
            Myxococcus xanthus is a bacterial micro-predator known for hunting other microbes in a wolf pack-like manner. Outer membrane vesicles (OMVs) are produced in large quantities by M. xanthus and have a highly organized structure in the extracellular milieu, sometimes occurring in chains that link neighboring cells within a biofilm. OMVs may be a vehicle for mediating wolf pack activity by delivering hydrolytic enzymes and antibiotics aimed at killing prey microbes. Here, both the protein and small molecule cargo of the OMV and membrane fractions of M. xanthus were characterized and compared. Our analysis indicates a number of proteins that are OMV-specific or OMV-enriched, including several with putative hydrolytic function. Secondary metabolite profiling of OMVs identifies 16 molecules, many associated with antibiotic activities. Several hydrolytic enzyme homologs were identified, including the protein encoded by MXAN_3564 (mepA), an M36 protease homolog. Genetic disruption of mepA leads to a significant reduction in extracellular protease activity suggesting MepA is part of the long-predicted (yet to date undetermined) extracellular protease suite of M. xanthus.
              Cover page: The lethal cargo of Myxococcus xanthus outer membrane vesicles
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