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Open Access Publications from the University of California

Scholarly Works (16 results)

  • Article
  • Peer Reviewed
UC San Diego Previously Published Works (2023)
Recent advances in cryo-electron microscopy have marked only the beginning of the potential of this technique. To bring structure into cell biology, the modality of cryo-electron tomography has fast developed into a bona fide in situ structural biology technique where structures are determined in their native environment, the cell. Nearly every step of the cryo-focused ion beam-assisted electron tomography (cryo-FIB-ET) workflow has been improved upon in the past decade, since the first windows were carved into cells, unveiling macromolecular networks in near-native conditions. By bridging structural and cell biology, cryo-FIB-ET is advancing our understanding of structure-function relationships in their native environment and becoming a tool for discovering new biology.
    Cover page: Bringing Structure to Cell Biology with Cryo-Electron Tomography.
    • Thesis
    • Peer Reviewed
    UC Berkeley Electronic Theses and Dissertations (2019)

    Autophagy (self-eating) is an essential process for cellular self-renewal. The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is central to autophagy initiation. The V-shaped architecture of the four-subunit version of PI3KC3-C1 consists of VPS34, VPS15, BECN1, and ATG14. Chapter 2 shows that a putative fifth subunit, NRBF2, is a tightly-bound component of the complex that profoundly affects its activity and architecture. NRBF2 enhances the lipid kinase activity of the catalytic subunit, VPS34. We used hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) and negative stain electron microscopy to map NRBF2 to the base of the V-shaped complex. NRBF2 interacts primarily with the N-termini of ATG14 and BECN1 and activates VPS34 through its N-terminal MIT domain.

    The ability of NRBF2 to activate the lipid kinase of VPS34 via an allosteric mechanism had not been determined. Chapter 3 shows precisely where the MIT domain of NRBF2 binds to PI3KC3-C1, that one copy of the MIT domain is insufficient for activation, and that full activation requires a second copy of the MIT domain. Dimeric NRBF2 cinches around the conformational flexible catalytic arm of the complex and stabilizes the lipid kinase domain of VPS34 and the putative kinase domain of VPS15.

    Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. Chapter 4 describes how a cryptic membrane binding site within BECN1 was identified. First, the interaction of PI3KC3 complex II and its ubiquitously expressed inhibitor, Rubicon, was mapped to the first β sheet of the BECN1 BARA domain by hydrogen-deuterium exchange and cryo-EM. The interaction determinants were confirmed in cell-based assays of PI3KC3 activity and autophagy. These data suggested that BARA β sheet-1 unfolds to directly engage the membrane. This mechanism was confirmed using protein engineering, giant unilamellar vesicle assays, and molecular simulations. Using this mechanism, a BECN1 β sheet-1-derived peptide activates both PI3KC3 complexes I and II, while HIV-1 Nef inhibits complex II. These data reveal how BECN1 switches on and off PI3KC3 binding to membranes. The observations explain how PI3KC3 inhibition by Rubicon, activation by autophagy-inducing BECN1 peptides, and inhibition by HIV-1 Nef, are mediated by the switchable ability of the BECN1 BARA domain to partially unfold and insert into membrane.

      Cover page: Turning Autophagy On and Off through the Class III PI-3 Kinase
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2017)
      Autophagy is the process of cellular self-eating by a double-membrane organelle, the autophagosome. A range of signaling processes converge on two protein complexes to initiate autophagy: the ULK1 (unc51-like autophagy activating kinase 1) protein kinase complex and the PI3KC3-C1 (class III phosphatidylinositol 3-kinase complex I) lipid kinase complex. Some 90% of the mass of these large protein complexes consists of noncatalytic domains and subunits, and the ULK1 complex has essential noncatalytic activities. Structural studies of these complexes have shed increasing light on the regulation of their catalytic and noncatalytic activities in autophagy initiation. The autophagosome is thought to nucleate from vesicles containing the integral membrane protein Atg9 (autophagy-related 9), COPII (coat protein complex II) vesicles, and possibly other sources. In the wake of reconstitution and super-resolution imaging studies, we are beginning to understand how the ULK1 and PI3KC3-C1 complexes might coordinate the nucleation and fusion of Atg9 and COPII vesicles at the start of autophagosome biogenesis.
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2019)
        Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol (PI) 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the proautophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryoelectron microscopy was used to visualize a 2-step PI3KC3-C1 activation pathway driven by NRFB2 MIT domain binding. Binding of a single NRBF2 MIT domain bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2019)
          Macroautophagy/autophagy is an evolutionarily conserved degradation system with fundamental biological functions. The activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complexes and the subsequent production of phosphatidylinositol 3-phosphate (PtdIns3P) are pivotal to autophagy. Using a combination of structural biology, biochemistry, and biophysics, we revealed how the non-catalytic subunit BECN1 serves as a membrane-binding switch in the regulation of PtdIns3K complexes and autophagy.
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2019)
            Macroautophagy/autophagy is an evolutionarily conserved degradation system with fundamental biological functions. The activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complexes and the subsequent production of phosphatidylinositol 3-phosphate (PtdIns3P) are pivotal to autophagy. Using a combination of structural biology, biochemistry, and biophysics, we revealed how the non-catalytic subunit BECN1 serves as a membrane-binding switch in the regulation of PtdIns3K complexes and autophagy.
              Cover page: The BARA necessities of PtdIns 3-kinase activation in autophagy
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2019)
              Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol (PI) 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the proautophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryoelectron microscopy was used to visualize a 2-step PI3KC3-C1 activation pathway driven by NRFB2 MIT domain binding. Binding of a single NRBF2 MIT domain bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.
                Cover page: Structural pathway for allosteric activation of the autophagic PI 3-kinase complex I
                • Article
                • Peer Reviewed
                UC Berkeley Previously Published Works (2020)
                The autophagy-initiating human ULK complex consists of the kinase ULK1/2, FIP200, ATG13, and ATG101. Hydrogen-deuterium exchange mass spectrometry was used to map their mutual interactions. The N-terminal 640 residues (NTD) of FIP200 interact with the C-terminal IDR of ATG13. Mutations in these regions abolish their interaction. Negative stain EM and multiangle light scattering showed that FIP200 is a dimer, while a single molecule each of the other subunits is present. The FIP200NTD is flexible in the absence of ATG13, but in its presence adopts the shape of the letter C ∼20 nm across. The ULK1 EAT domain interacts loosely with the NTD dimer, while the ATG13:ATG101 HORMA dimer does not contact the NTD. Cryo-EM of the NTD dimer revealed a structural similarity to the scaffold domain of TBK1, suggesting an evolutionary similarity between the autophagy-initiating TBK1 kinase and the ULK1 kinase complex.
                  Cover page: ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer
                  • Article
                  • Peer Reviewed
                  UC Berkeley Previously Published Works (2020)
                  The autophagy-initiating human ULK complex consists of the kinase ULK1/2, FIP200, ATG13, and ATG101. Hydrogen-deuterium exchange mass spectrometry was used to map their mutual interactions. The N-terminal 640 residues (NTD) of FIP200 interact with the C-terminal IDR of ATG13. Mutations in these regions abolish their interaction. Negative stain EM and multiangle light scattering showed that FIP200 is a dimer, while a single molecule each of the other subunits is present. The FIP200NTD is flexible in the absence of ATG13, but in its presence adopts the shape of the letter C ∼20 nm across. The ULK1 EAT domain interacts loosely with the NTD dimer, while the ATG13:ATG101 HORMA dimer does not contact the NTD. Cryo-EM of the NTD dimer revealed a structural similarity to the scaffold domain of TBK1, suggesting an evolutionary similarity between the autophagy-initiating TBK1 kinase and the ULK1 kinase complex.
                    Cover page: ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer
                    • Article
                    • Peer Reviewed
                    UC Berkeley Previously Published Works (2015)
                    Diverse organisms capable of surviving desiccation, termed anhydrobiotes, include species from bacteria, yeast, plants, and invertebrates. However, most organisms are sensitive to desiccation, likely due to an assortment of different stresses such as protein misfolding and aggregation, hyperosmotic stress, membrane fracturing, and changes in cell volume and shape leading to an overcrowded cytoplasm and metabolic arrest. The exact stress(es) that cause lethality in desiccation-sensitive organisms and how the lethal stresses are mitigated in desiccation-tolerant organisms remain poorly understood. The presence of trehalose in anhydrobiotes has been strongly correlated with desiccation tolerance. In the yeast Saccharomyces cerevisiae, trehalose is essential for survival after long-term desiccation. Here, we establish that the elevation of intracellular trehalose in dividing yeast by its import from the media converts yeast from extreme desiccation sensitivity to a high level of desiccation tolerance. This trehalose-induced tolerance is independent of utilization of trehalose as an energy source, de novo synthesis of other stress effectors, or the metabolic effects of trehalose biosynthetic intermediates, indicating that a chemical property of trehalose is directly responsible for desiccation tolerance. Finally, we demonstrate that elevated intracellular maltose can also make dividing yeast tolerant to short-term desiccation, indicating that other disaccharides have stress effector activity. However, trehalose is much more effective than maltose at conferring tolerance to long-term desiccation. The effectiveness and sufficiency of trehalose as an antagonizer of desiccation-induced damage in yeast emphasizes its potential to confer desiccation tolerance to otherwise sensitive organisms.
                      Cover page: Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae
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