1. Thursday 9/4 2014 Mike Mueckler mmueckler@wustl.edu

2. Intracellular Targeting of Nascent Polypeptides

3. Figure 14-10 Molecular Biology of the Cell (© Garland Science 2008) Mitochondria are the Sites of Oxidative ATP Production SugarsTriglycerides

4. Mitochondrial Biogenesis Mitochondria contain their own genome and protein synthetic machinery (tRNAs, mRNAs, ribosomes, initiation and elongation factors, etc.) Mitochondria are comprised of hundreds of distinct proteins, only a handful of which are encoded in the mitochondrial genome (varies by species) Most mitochondrial proteins are encoded in nuclear DNA, synthesized in the cytosol, and imported post-translationally into the organelle

5. Mitochondria Possess 4 Subcompartments

6. Use of in vitro Systems to Elucidate Mitochondrial Import Mechanisms

7. Figure 12-23 Molecular Biology of the Cell (© Garland Science 2008) Proteins are Incorporated Into Mitochondria Via Several Different Routes

8. Targeting to the Matrix Requires an N- Terminal Import Sequence

9. Figure 12-22 Molecular Biology of the Cell (© Garland Science 2008) N-terminal Import Sequences Form Amphipathic  Helices that Interact with the Tom20/22 Receptor Hydrophobic cleft

10. Protein Import into the Matrix Requires Passage Through Two Separate Membrane Translocons

11. Proteins Traverse the TOM and TIM Translocons in an Unfolded State

12. Translocation into the Matrix Occurs at Zones of Adhesion

13. Figure 12-26 Molecular Biology of the Cell (© Garland Science 2008) Protein Import into the Matrix Requires ATP Hydrolysis and an Intact Proton Gradient Across the Inner Membrane

14. Targeting to the Inner Membrane Occurs Via 3 Distinct Routes Oxa1-MediatedStop-Transfer-MediatedTom70/Tim22/54-Mediated Multi-Pass Proteins Single-Pass Proteins Cytochrome oxidase subunit CoxVa ATP Synthase Subunit 9 ADP/ATP Antiporter

15. Cytochrome B2Cytochrome c Heme Lyase Targeting to the Intermembranous Space Occurs Via Two Distinct Pathways Direct Delivery IM Space Protease

16. Figure 12-27 Molecular Biology of the Cell (© Garland Science 2008) Targeting to the Outer Membrane Via the SAM Protein Complex ( S orting and A ssembly Machinery) (  -Barrell)

17. Figure 12-8 Molecular Biology of the Cell (© Garland Science 2008) Nuclear Transport Bidirectional Single Large Pore Complex Spans 2 lipid bilayers Nuclear Pores much larger than other translocons

18. Figure 12-9c Molecular Biology of the Cell (© Garland Science 2008) EM of Transverse Section Showing a Side-View through two NPCs

19. Figure 12-9b Molecular Biology of the Cell (© Garland Science 2008) Scanning EM of NPCs as Viewed from the Nucleoplasm

20. Figure 12-9a Molecular Biology of the Cell (© Garland Science 2008) Structure of a Nuclear Pore Complex

21. Figure 12-10 Molecular Biology of the Cell (© Garland Science 2008) Gated Diffusion Barrier Model of Nuclear Transport Meshwork of disordered protein domains containing FG repeats

22. Figure 12-11 Molecular Biology of the Cell (© Garland Science 2008) Nuclear Import Signals are Highly Diverse in Sequence Bind to distinct nuclear import receptors Can be anywhere in the protein sequence but probably reside on surface patches Some are not yet identified

23. Figure 12-12 Molecular Biology of the Cell (© Garland Science 2008) Gold Particles Coated with Peptides Containing a NLS Traverse NPCs Proteins do not have to be unfolded before they traverse the nuclear pore

24. Figure 12-13 Molecular Biology of the Cell (© Garland Science 2008) Nuclear Import and Export Sequences are Recognized by Different Members of the Same Receptor Family (Keryopherins)

25. Figure 12-14 Molecular Biology of the Cell (© Garland Science 2008) Directionality is Conferred on Nuclear Transport by a Gradient of Ran-GDP/GTP Across the Nuclear Envelope

26. Figure 12-15 Molecular Biology of the Cell (© Garland Science 2008) Nuclear Import and Export Operate Via Reciprocal Use of the Ran-GDP/GTP Concentration Gradient