Presentation on theme: "MCB Exam I Review Dr. Hanson and Morley Ji Park. Fate of newly synthesized glycoproteins in the ER I Path when nascent protein folds efﬁciently (green."— Presentation transcript:
Fate of newly synthesized glycoproteins in the ER I Path when nascent protein folds efﬁciently (green arrows) Players – OST = oligosaccharyl transferase – GI, GII = glucosidase I and II – Cnx/Crt = Calnexin and Calreticulin, lectin chaperones – ERp57 = oxidoreductase – ERMan1 = ER mannosidase 1 – ERGIC53, ERGL, VIP36 = lectins that facilitate ER exit Increases solubility glucose mannose
Fate of newly synthesized glycoproteins in the ER II Path when nascent protein goes through folding intermediates (orange arrows) Players – UGT1 (a.k.a. UGGT) = UDP- glucose–glycoprotein glucosyltransferase, recognizes nearly native proteins, acting as conformational sensor – Reglucosylated protein goes through Cnx/Crt cycle for another round " – GII removes glucose to try again and pass QC of UGT1 – BiP = hsc70 chaperone that recognizes exposed hydrophobic sequences on misfolded proteins
Fate of newly synthesized glycoproteins in the ER III Folding-defective proteins need to be degraded - transported out of the ER for degradation How do proteins avoid futile cycles? – UGT1 does not recognize fatally misfolded proteins and won t reglucosylate them for binding to Cnx/Crt – Resident mannosidases will trim mannose residues - protein can no longer be glucosylated and bind to Cnx/ Crt – BiP binds hydrophobic regions – Mannosidase trimmed glycans recognized by OS9 associated with ubiquitination machinery Leads to kinetic competition between folding and degradation of newly synthesized glycoproteins " " Slow
The UPR in yeast Ire1=inositol-requiring protein-1, ER-localized transmembrane kinase and site speciﬁc endoribonuclease Ire1 is maintained in inactive state by binding to BiP. Removal of BiP (by binding to misfolded proteins) leads to Ire1 activation Ire1 activation triggers splicing of intron in mRNA encoding Hac1, a dedicated UPR transcriptional activator Hac1 then binds to UPRE elements to selectively upregulate gene expression of targets that will help alleviate the overload of misfolded proteins
Unfolded Protein Response in Metazoans Three branches Cells respond to ER stress by: – Reducing the protein load that enters the ER Transient Decreased protein synthesis and translocation – Increase ER capacity to handle unfolded proteins Longer term adaptation Transcriptional activation of UPR target genes – Cell death Induced if the ﬁrst two mechanisms fail to restore homeostasis
NSF provided tool to identify additional components… including the membrane fusion machinery SNAPs - soluble NSF attachment proteins" – NSF and SNAPs are general factors required for vesicular transport. " SNAREs - SNAP Receptors " – Large family of proteins; speciﬁc SNAREs for each compartment; mediate membrane fusion"
Discovery that clostridial toxins cleave SNAREs supports role in fusion Toxins found to be metalloproteases Toxins quickly inhibit synaptic vesicle exocytosis Therefore targets must be important for neurosecretion Comparison of treated vs. non-treated synaptosomes shows cleavage of VAMP/synaptobrevin Injecting peptides encompassing cleavage site in VAMP/synaptobrevin inhibit action of tetanus toxin VAMP/synaptobrevin
Transport through the Golgi: anterograde, retrograde or both? Two models to debated over many years –Stable Compartments connected by vesicle trafﬁc: secretory cargo (large and small) moves through these by a vesicle-mediated anterograde process –Cisternal Maturation: VTCs fuse into an ERGIC (ER- Golgi intermediate compartment). This matures into the cis-Golgi by gain of Golgi proteins and removal of VTC proteins via COPI vesicles that move in a retrograde direction. Cis-Golgi then matures to medial Golgi by similar mechanism, etc. "
Problems with lysosomal function: I-cell disease Caused by deficiency in GlcNAc- phosphotransferase, lysosomal enzymes therefore lack M6PR tag Leads to secretion of multiple lysosomal enzymes, cells become vacuolated and contain dense inclusion bodies Clinical manifestations: severe skeletal and neurological problems, retardation of grow and development, death by 5 yrs
Clathrin mediated endocytosis * predominant endocytic pathway * membrane and fluid uptake, responsible for most receptor mediated endocytosis * 2-3% of cell surface occupied by clathrin coated pits * lifetime of coated pit estimated to be ~1 minute before pinching off as coated vesicle clathrin lattice clathrin molecule clathrin coated pit AP-2 adaptor
Clathrin coated vesicle cycle Adaptor protein(s) bind to membranes and cargo Adaptor protein(s) recruit clathrin, create nascent vesicle Clathrin and adaptor proteins are sufficient to form lattices and buds on liposomes, but cooperate with other accessory proteins in vivo Accessory proteins regulate coat assembly, membrane fission, and clathrin coat disassembly
Caveolar endocytosis Minor pathway compared to clathrin mediated endocytosis Internalizes membranes enriched in lipid rafts Pathway used by GPI-anchored proteins, toxins, viruses Internalized caveolae travel to caveosome, ER, Golgi, endosome Pathway requires dynamin, actin, and probably others Key difference from clathrin pathway is that caveolar coat does not disassemble, instead contents diffuse out or dissociate
LDL (low density lipoprotein particle) receptor: receptor recycles, cargo is degraded Cell gets amino acids, cholesterol,fatty acids from degraded LDL
Transferrin receptor: receptor and transferrin recycle, Fe 3+ internalized * Prototypical recycling receptor * t 1/2 for recycling ~ 16 min * similar to kinetics of bulk lipid recycling * receptor recycles 100+ x during lifetime
EGF receptor: receptor, EGF degraded Accumulates in coated pits only after ligand binding Internalization requires active kinase domain Receptor and ligand both delivered to lysosomes & degraded Results in receptor down- regulation
Generation of actin filaments: getting from G to F Dominguez, Crit Rev Biochem Mol Biol 2009. Nucleation: energetically unfavorable and a key step in generating filament Terms to know Elongation: ATP adds faster than ADP; barbed > pointed Depolymerization: ADP more likely to depolymerize; pointed > barbed Critical concentration: conc of G-actin at which poly = depoly (conc at which equilibrium between G-actin and F-actin exists) Treadmilling: barbed polymerizes while pointed depolymerizes
Generation of actin filaments: layers of control Nucleotide binding – profilin (exchange factor) Layers of control that enable rapid remodeling of actin cytoskeleton Nucleators – Arp2/3 complex, formins Filament cleavage (increases number of ends, shortens filaments) – gelsolin Filament capping to prevent elongation – capping protein Creation of larger order structures (building with F-actin) – fimbrin G-actin sequestration (alters concentration) – cofilin (promotes hydrolysis) The Cell: A Molecular Approach 2 nd ed
Profilin 13 – 19 kDa Catalyze exchange of ADP ATP Prevents addition to pointed end of filament Prevents nucleation Permits binding to barbed end Contains a proline-rich sequence (PRS) that enables binding to other actin-regulatory proteins (cooperates with formins) barbed face pointed face profilin PRS Allergen! IgE against profilin in pollen, plant food, natural rubber latex allergies Four profilin isoforms: profilin-1 ubiquitously expressed, 2/3/4 have more specific patterns of expression Pfn1 -/- mice die before blastocyst stage Xue and Robinson, Eur J Cell Biol 2013.
Actin Depolymerizing Factor (ADF)/Cofilin family pointed barbed Cofilin binds to F-actin, promotes ATP ADP hydrolysis pointed barbed Effect of ADF/cofilin is pH- and concentration-dependent Low conc: severs F-actin, promotes depolymerization of pointed end High conc: increases polymerization by nucleating new filaments Cofilin binds to G-actin, inhibits ADP ATP exchange 19 kDa, ubiquitous in mammalian cells CFN1 (non-muscle), CFN2 (muscle), ADF CFN1 -/- mice embryonic lethal Hild et al. Eur J Cell Biol 2013.
Capping protein (CP) Works with Arp2/3 to generate branched networks at leading edge of migrating cells CP null in Drosophila lethal in early larval stage / heterodimer, each subunit 30 kDa Binds to barbed end of F-actin Prevents addition of monomers, thus “capping” Kim, Cooper and Sept, J Mol Biol, 2010. CP knockdown loss of lamellipodia barbed pointed
Actin-related protein (Arp) 2/3 complex Binds to the side of F-actin; generates novel filaments in branching pattern (70 o ) Complex of seven different proteins, including Arp2 and Arp3 Activated by upstream regulators (Nucleation Promoting Factors): WASP (Wiskott-Aldrich Syndrome Protein) being the most famous Binding of actin monomer to Arp2/3 actin-like trimer (sufficient for nucleation) Boczkowska et al, Structure, 2008.
Formins Schoneichen and Geyer, Biochim Biophys Acta, 2010. actin nucleation and elongation pointed
Gelsolin Multiple functions: severs F-actin, caps barbed end, can enable filament disassembly from pointed end Can also nucleate filaments in polymerizing conditions, though whether happens in cells in vivo is controversial Six-domain structure, 755 amino acids Activates gel sol(uble) transformation of F-actin Regulated by calcium, phosphoinositide binding McGough et al, FEBS Letters, 2003. G-actin bindingF-actin binding G-actin binding (Ca 2+ ) Gsn -/- mice on Balb/C background = embryonic lethal, possibly due to defective RBC maturation. Disappointingly mild phenotype in B6 mice.
Ras-related proteins. GTP-bound = active, GDP-bound = inactive Small GTP-binding proteins: Rac, Rho, cdc42 Rac: lamellipodia; Rho: stress fiber formation; cdc42: filopodia Guanine nucleotide exchange factors (GNEFs): catalyzed exchange of GDP for GTP (upstream activators of Rac/Rho) GTP-ase-activating proteins (GAPs): catalyze GTP GDP RAC GDP RAC GTP GNEFs (Vav) GAPs
WASP: Closed and open conformations Matalon et al, Immunol Rev, 2013