Tuesday 9/6/2018 Mike Mueckler

Transcription

1 Tuesday 9/6/2018 Mike Mueckler

2 Intracellular Targeting of Nascent Polypeptides

3 Mitochondria are the Sites of Oxidative ATP Production Sugars Triglycerides Figure Molecular Biology of the Cell ( Garland Science 2008)

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 Proteins are Incorporated Into Mitochondria Via Several Different Routes Figure Molecular Biology of the Cell ( Garland Science 2008)

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

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

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

14 Targeting to the Inner Membrane Occurs Via 3 Distinct Routes Stop-Transfer-Mediated Oxa1-Mediated Tom70/Tim22/54-Mediated Single-Pass Proteins Cytochrome oxidase subunit CoxVa ATP Synthase Subunit 9 Multi-Pass Proteins ADP/ATP Antiporter

15 Targeting to the Intermembranous Space Occurs Via Two Distinct Pathways IM Space Protease Direct Delivery Cytochrome B2 Cytochrome c Heme Lyase

16 Targeting to the Outer Membrane Via the SAM Protein Complex (Sorting and Assembly Machinery) (β-barrell) Figure Molecular Biology of the Cell ( Garland Science 2008)

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

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

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

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

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

22 Nuclear Import Signals are Highly Diverse in Sequence Figure Molecular Biology of the Cell ( Garland Science 2008) 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 Gold Particles Coated with Peptides Containing a NLS Traverse NPCs Proteins do not have to be unfolded before they traverse the nuclear pore Figure Molecular Biology of the Cell ( Garland Science 2008)

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

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

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

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