Promoting t-SNARE Binary Complex Assembly and membrane Fusion by the Exocyst Protein Sec3
Peng Yue
A DISSERTATION in Biology
Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
2015
Supervisor of Dissertation
______________________
Wei Guo, Professor of Biology
Graduate Group Chairperson
______________________
Michael Lampson, Associate Professor of Biology
Dissertation Committee
Fevzi Daldal, Professor of Biology
Tatyana Svitkina, Professor of Biology
Erfei Bi, Professor of Cell and Developmental Biology
Claudio G. Giradudo, Assistant Professor of Pathology and Laboratory Medicine
James Shorter, Associate Professor of Biochemistry and Biophysics
ACKNOWLEDGMENT (optional)
ABSTRACT
Promoting t-SNARE Binary Complex Assembly and membrane Fusion by the Exocyst Protein Sec3
Exocyst is an octameric protein complex that mediates the initial contact between secretory vesicles and the plasma membrane. The final fusion event is mediated by SNAREs (Soluble N-ethylmaleimide Sensitive Factor Attachment Protein Receptor), proteins residing on both vesicles and the PM. The assembly of the SNARE complex drives membrane fusion. SNARE mediated fusion at the plasma membrane in yeast begins with the formation of a binary t SNARE complex composed of Sso1/2 and Sec9 followed by its binding to the v SNARE protein Snc1/2. It was previously shown that SNARE assembly is
One of the main manufacturing facilities within a cell. Consists of rough ER and smooth ER, they are physically connected but differ in structure. Consist of membrane-enclosed tubes and sacs within the cytoplasm. Rough ER, named for the ribosomes attached to its surface, make membrane and secretory proteins. One function of rough ER is to produce new membrane. Some products by rough ER are dispatched to other locations by transport vesicles.
-Sarcoplasmic Reticulum (SR) then releases Calcium which binds to troponin in the thin filament, exposing myosin-binding sites;
Membranes can allow or exclude various molecules, and because of selective transport systems (active mediated transport), they can move molecules in and out of the space. Membrane channels, or “gates,” can open and close depending on the circumstances of the first messenger. Binding of an extracellular messenger to a dual receptor/channel brings about a quick
“So, this is Mike the Mitochondria and he is the main power source. His specialty is cellular respiration which is when he breaks down food molecules to make ATP.”
The body has two faces, the cis face which fuses with incoming transport vesicles, and the trans face which excretes the secretory vesicles. The cis face fuses with vesicles coming from the ER effectively from many directions due to its convex shape, whereas the concave trans face can direct the secretory vesicles to their destination. When fusing with the cis face, the transport vesicles release their proteins to be absorbed for modification. Each cisternal layer of the Golgi body holds different enzymes which each modify the passing proteins in separate ways. Between the layers the proteins are moved through the gaps by small vesicles. When a protein has been modified correctly, it leaves the Golgi body via secretory vesicles which then carry the modified proteins to the cell membrane or another organelle. The proteins that are transported to the cell membrane are either excreted from the cell, or absorbed into the membrane to aid with its function. Some of the secretory vesicles which hold hydrolytic enzymes stay within the cytoplasm and function as lysosomes.
The human body is made up of 100 trillion cells. All cells have the same basic structure (see figure 1) however some cells are specialised to suit a specific purpose. An example of this is the pancreatic beta cells found in the islets of Langerhans. These cells are specialised to synthesise the protein insulin that is involved in the metabolism of glucose in the cells. (Layden, 2010)
Chaperones are proteins that ensure the correct folding of the CFTR within the endoplasmic reticulum. Hsp70 is an important cytosolic chaperone that complexes with CFTR and reduces aggregation [5]. The CFTR passes through the endoplasmic reticulum-associated degradation (ERAD) after folding in the ER. This quality control system involves the ubiquitin proteasome system (UPS) for which CFTR is a substrate [16]. If a protein is molded and targeted for degradation, then ubiquitin will covalently attach to lysine residues on the CFTR. Three enzymes are required for the process of ubiquitylation: E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin protein ligases. E1 enzymes are activated through hydrolysis of ATP, which creates an activated ubiquitin that is transferred to an E2 active site. The activated ubiquitin is then covalently bound to a lysine on the protein by an E3 ligase. A polyubiquitin chain is then formed as ubiquitin molecules link together, and if there are four or more then the misfolded CFTR chain is removed form the ER membrane and targeted for degradation by the 26S proteasome
Numerous cells in the body utilize exocytosis to discharge chemicals or different proteins that demonstration in different zones of the body, or to discharge particles that assistance cells speak with each other. For example, bunches of α-and β-cells in the islets of Langerhans in the pancreas emit the hormones glucagon and insulin, individually. These catalysts manage glucose levels all through the body. As the level of glucose ascends in the blood, the β-cells are fortified to deliver and discharge more insulin by exocytosis. At the point when insulin ties to liver or muscle, it animates take-up of glucose by those cells. Exocytosis from different cells in the pancreas additionally discharges stomach related chemicals into the gut.
SPI-1 is the best characterized of the SPIs. It is approximately 40 kb in size. SPI-1 is flanked by fhlA and mutS and has G-C content about 42%. SPI-1 contains at least 29 genes, encoding various components protein of a type III secretion system. SPI-1 encodes several effector proteins which mostly involved in invasion of epithelial cells by mediating actin cytoskeletal rearrangement and hence internalization of the bacteria. These effectors were translocated into the host cell by type III secretion system (T3SS), which also encoded on SPI-1. The prg/org and inv/spa operons encode d for the needle complex, whereas the sic/sip operons encoded for the effector proteins and the translocon (SipBCD), a pore-forming structure that embeded in the host cell membrane. In addition, several chaperones are also encoded within SPI-1 and these chaperones protect SPI-1-related proteins
Proteins, carbohydrates, lipids, and nucleic acids are all essential macromolecules for basic biological functions within an organism’s body, and other everyday applications. They are used in chemical reactions to achieve many results, such as energy storage, DNA coding, waste management, and a number of other uses. However, most of the chemical reactions involving macromolecules rely heavily on the shape and structure of the molecule; such as the amount of monomers in the macromolecule. The process of joining macromolecule monomers together is a chemical reaction called dehydration synthesis, or also known as a condensation reaction. The functional groups of a macromolecule are reacted together to form various types of bonds, and produce water
Capturing O-GlcNAc mediated protein-protein interactions is challenging due to the low-affinity, sub-stoichiometric and transient nature of the modification. Using a synthetic O-GlcNAc analog containing a diazirine photocrosslinking moiety (GlcNDAz), we are able to label native O-GlcNAcylated substrates in live cells and crosslink them to nearby glycosylation-mediated binding partners upon UV treatment 13. In experiments performed by Abhisheck Chhetri, Tim Smith and Thomas Meister, the Boyce lab showed that endogenous and recombinant Myc-6xHis-tagged Sec24C exhibit GlcNDAz- and UV-dependent crosslinking (Figure 2). Two other Sec24 paralogs, Sec24A and Sec24B, also show GlcNDAz-dependent crosslinking. Additionally, through collaboration with Duke’s Proteomics and Metabolomics Core Facility, I have identified three O-GlcNAcylated residues on Sec24C: Ser97, Thr98, and Thr201. A large amount of recombinant Myc-6xHis-tagged Sec24C was purified by Myc immunoprecipitation (IP) and Ni-NTA purification from HEK 293T cells. Should include more detail about how you did this, especially since it’s currently the only experiment in the Preliminary Data section that
Four ways that large molecules and substances are transported across a membrane include phagocytosis, pinocytosis, receptor-mediated endocytosis and receptor proteins. During phagocytosis, the cell engulfs a particle by wrapping pseudopodia around the particle and packing the particle within the food vacuole (membranous sac). Once the food vacuole integrates with a lysosome (w/ hydrolytic enzyme), the particle will be digested. The second way is pinocytosis, in which the cell takes in “droplets” of extracellular fluid and packs it into tiny vesicles; after this, the tiny vesicles are then transported into the cell because the molecules dissolved in the droplets are the main factors that the cells need. The third process is known as receptor-mediated endocytosis which the cells takes in large quantities of specific substances of all concentration in the Extracellular (EC) fluid; the membranes of the cell vesicle are embedded with proteins that has certain receptor sites that are exposed to the EC fluid in which ligand binds to. Then, the last step is that the receptor proteins cluster in regions of the membrane known as coated pits which contain fuzzy layer of coat proteins on the exterior; then, each coated pit forms a vesicle which contains the ligand molecules and after the ingested material is released from the vesicle, the vesicles then recycle the receptors to the plasma
Exosomes are membranous vesicles enclosed in lipid bilayers with diameter of 30-100nm and are responsible in carrying cytoplasmic proteins such as Rab proteins and actin-binding proteins and signal transduction molecules such as G-proteins and protein kinases, as well as some heat-shock proteins like Hsp70 and tetraspanins like CD9 [5-30]. Exosomal proteins can directly represent the specific profiles of their cells of origin, for
The endoplasmic reticulum (ER) is an essential organelle that is a major place for the biogenesis of cellular components including proteins, lipids, and carbohydrates and internal calcium storage. ER is primarily responsible for protein translocation, protein folding and protein post modification. Proper folding of protein in the ER is accomplished with the aid of ER resident proteins or enzymes such as chaperones. Binding of chaperones to
Overexpression of the HEPN domain in HeLa cells following three hours of starvation indicates that sacsin plays a key role in lysosomal transport due to the reduced efficiency of perinuclear lysosomal clustering. HEPN’s property of dimerizing may be disrupting the function of with full-length sacsin by preventing endogenous sacsin from dimerizing within the cell. Thus, dimerization may be essential to sacsin’s function, particularly for binding JIP3, which is necessary for lysosomal transport7. HATPase 3 overexpression may be occupying JIP3 binding, but was not found to disrupt lysosomal localization following starvation, perhaps due to the truncated size of this deletion construct.