October 26, Lecture Readings. Vesicular Trafficking, Secretory Pathway, HIV Assembly and Exit from Cell

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1 October 26, 2006 Vesicular Trafficking, Secretory Pathway, HIV Assembly and Exit from Cell 1. Secretory pathway a. Formation of coated vesicles b. SNAREs and vesicle targeting 2. Membrane fusion a. SNAREs and vesicle fusion 3. Retrograde trafficking a. Steady-state 4. Functions of the Golgi a. Glycosylation b. Protein sorting 5. Viral assembly and exit from the cell Lecture Readings Alberts (cont.) 1

2 Challenges in Vesicular Transport I 1) How is membrane deformed to form a vesicle? 2) How are the correct cargoes enriched in the vesicle and the proteins that should remain in the donor compartment excluded? 3) How do vesicles know what compartment to fuse with? 4) What catalyzes the fusion process? 3 Major Types of Coated Vesicles 2

3 SNAREs and Targeting Vesicles coat protein SNAREs Promote Membrane Fusion v-snare vesicle SQUEEZE OUT WATER FORM STALK target t-snare HEMI- FUSION FUSION 3

4 Challenges in Vesicular Transport II 1) How does the cell maintain organelles and the plasma membrane at a constant size if vesicles are constantly moving lipids from compartments to the plasma membrane? 2) How does the cell ensure that proteins which should remain in a compartment do so, even if the process of cargo selection is not perfect? Secretory Pathway: Retrograde Traffic nuclear envelope endoplasmic reticulum lysosome late endosome plasma membrane early endosome CYTOSOL cisternae Golgi apparatus secretory vesicle 4

5 Equilibrium vs. Steady-State equilibrium steady-state Golgi Apparatus vesicles coming from ER vesicles leaving Golgi 5

6 Viral Entry and Integration reverse transcriptase plasma membrane RNA-DNA hybrid DNA chromosomal DNA viral RNA NUCLEUS Viral Assembly and Exit from the Cell gp120 and gp41 plasma membrane viral capsid viral genome mrna HIV viral proteins ER, Golgi, secretory pathway gp120 and gp41 virus budding gag polyprotein 6

7 Summary of Main Points Vesicles bud as coated vesicles containing a protein coat that plays a role in membrane deformation and cargo selection SNAREs play an important role in targeting vesicles to the correct compartment SNAREs play an important role in overcoming the energy barrier to membrane fusion; they function in a manner analogous to gp41 Retrograde, or reverse, traffic in the secretory pathway ensures that resident proteins remain in their compartments and that cells maintain organelles at a constant size; trafficking in the secretory pathway is at steady-state Proteins are sorted to their destinations in the Golgi and carbohydrates are further processed HIV assembles viral particles at the plasma membrane and buds from the cell; the viral membrane is derived from the host cell plasma membrane 7

8 1a OCTOBER 26, 2006 ROBERT A. LUE Nucleic acids in action: Transcription and the programming of the cell 1. Sustained HIV replication destroys the human immune system a) HIV targets key cells of the immune system b) Timecourse of untreated HIV infection 2. The principle of cellular cooption 3. From DNA to RNA: Transcription 4. HIV proviral transcription Lecture Readings Alberts: pp , McMurry: pp

9 HIV targets key cells of the immune system Innate immune responses are rapid and nonspecific Adaptive immune responses are slow and specific to particular pathogens Vertebrate innate immune responses activate adaptive immunity CD4 + T cells support adaptive immunity by activating other immune cells Helper T cells Macrophages destroy pathogens (innate) and present pathogen components for T cell recognition (adaptive) Timecourse of untreated HIV infection

10 Nucleic acids in action: Transcription and the programming of the cell 1. Sustained HIV replication destroys the human immune system 2. The principle of cellular cooption a) Cells have much to offer pathogens b) Listeria: an example of protein cooption 3. From DNA to RNA: Transcription 4. HIV proviral transcription Lecture Readings Alberts: pp , McMurry: pp Cells have much to offer pathogens Cells contain essential resources: Functional macromolecules and The directed synthesis of macromolecules

11 Cells have much to offer pathogens Cells contain essential resources: Functional macromolecules and The directed synthesis of macromolecules Example of a key protein function Protein monomers of Actin polymerize into filaments in the cytoplasm Key component of the cell cytoskeleton Listeria: an example of protein cooption Example: Listeria monocytogenes Bacteria that cause food poisoning Move in the cytosol of infected cells >10!m/min. Induces the polymerization of actin monomers at one end of the bacterium Bacterial protein (ActA) recruits actin monomers to one end of the bacterium Coopts existing cellular resources by direct binding

12 Listeria: an example of protein cooption ActA coated microspheres Polystyrene microspheres (1!m dia.) are asymmetrically coated with ActA and incubated in a cytoplasmic extract Listeria-infected cell L. Cameron & J.A. Theriot Nucleic acids in action: Transcription and the programming of the cell 1. Sustained HIV replication destroys the human immune system 2. The principle of cellular cooption 3. From DNA to RNA: Transcription a) Overview of intracellular information transfer b) RNA polymerase transcribes RNA from a DNA template c) Bacterial transcription as a model d) The starting and stopping of transcription in bacteria e) Eukaryotic transcription requires multiple initiation factors 4. HIV proviral transcription Lecture Readings Alberts: pp , McMurry: pp

13 Overview of intracellular information transfer Transcription of DNA into complementary RNA RNA polymerase transcribes specific regions of DNA into complementary RNA RNA Pol unwinds the DNA helix Catalyzes the polymerization of RNA using DNA as a template A transient DNA-RNA helix is formed The hybrid helix is disrupted to allow rewinding of the DNA

14 Bacterial RNA transcription Sigma factor subunit of RNA Pol recognizes the promoter sequence RNA is synthesized in the 5-3 direction Termination sequence halts RNA pol and induces release Starting and Stopping transcription in bacteria Specific DNA sequences define the beginning and end of a transcriptional unit Promoter sequences are asymmetric -- serves to orient the RNA polymerase Regulatory proteins affect the rate of initiation at the promoter

15 Starting and Stopping transcription in bacteria Terminator sequences induce the release the DNA template and RNA product The release process is facilitated by the formation of a RNA hairpin structure Eukaryotic RNA polymerases Eukaryotic cells have three RNA polymerase enzymes RNA pol II RNA polymerase I transcribes genes encoding ribosomal RNAs RNA polymerase II transcribes genes encoding proteins RNA polymerase III transcribes genes encoding transfer RNAs, ribosomal RNAs, and small structural RNAs

16 Eukaryotic RNA polymerase II Eukaryotic transcription requires multiple initiation factors Eukaryotic RNA polymerase II requires multiple transcription factors (TFs) for initiation General transcription factors assemble on all promoter sequences The TATA box (sequence) of the promoter binds the first TF Other TFs assemble at the promoter together with RNA Pol II A specific TF in the complex adds phosphate groups to RNA Pol II Addition of phosphate alters the activity of RNA Pol II, facilitating the completion of transcription

17 Nucleic acids in action: Transcription and the programming of the cell 1. Sustained HIV replication destroys the human immune system 2. The principle of cellular cooption 3. From DNA to RNA: Transcription 4. HIV proviral transcription a) Reverse transcription b) Map of the HIV genome c) HIV Tat protein enhances proviral transcription d) Tat recruits host cell proteins that alter the activity of RNA polymerase II Lecture Readings Alberts: pp , McMurry: pp HIV Reverse Transcription

18 HIV Reverse Transcriptase RT reverse transcribes the single-stranded RNA genome into double-stranded DNA before integration 1) RNA to DNA (RNA-dependent DNA polymerase) 2) Digests RNA from RNA-DNA hybrid (RNAse function) 3) Make DNA from DNA, i.e. double-stranded (DNA-dependent DNA polymerase) Nucleoside analog RT inhibitors AZT is an analog of thymidine Lacks the 3 -OH required for the addition of the next nucleoside triphosphate Inhibits reverse transcription by competing for the nucleoside substrate and terminating chain polymerization

19 Mechanism of AZT inhibition