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TARGETING OF NASCENT POLYPEPTIDES OUTSIDE OF THE SECRETORY PATHWAY
Thursday 9/4 2014Mike Mueckler
Intracellular Targeting of Nascent Polypeptides
Figure 14-10 Molecular Biology of the Cell (© Garland Science 2008)
Mitochondria are the Sites of Oxidative ATP Production
Sugars Triglycerides
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
Mitochondria Possess 4 Subcompartments
Use of in vitro Systems to Elucidate Mitochondrial Import Mechanisms
Figure 12-23 Molecular Biology of the Cell (© Garland Science 2008)
Proteins are Incorporated Into Mitochondria Via Several Different Routes
Targeting to the Matrix Requires an N-Terminal Import Sequence
Figure 12-22 Molecular Biology of the Cell (© Garland Science 2008)
N-terminal Import Sequences Form Amphipathic a Helices that Interact with the Tom20/22 Receptor
Hydrophobic cleft
Protein Import into the Matrix Requires Passage ThroughTwo Separate Membrane Translocons
Proteins Traverse the TOM and TIM Translocons in an Unfolded State
Translocation into the Matrix Occurs at Zones of Adhesion
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
Targeting to the Inner Membrane Occurs Via 3 Distinct Routes
Oxa1-MediatedStop-Transfer-Mediated Tom70/Tim22/54-Mediated
Multi-Pass ProteinsSingle-Pass Proteins
Cytochrome oxidase subunit CoxVa
ATP Synthase Subunit 9
ADP/ATP Antiporter
Cytochrome B2 Cytochrome c Heme Lyase
Targeting to the Intermembranous Space Occurs Via Two Distinct Pathways
Direct DeliveryIM Space Protease
Figure 12-27 Molecular Biology of the Cell (© Garland Science 2008)
Targeting to the Outer Membrane Via the SAM Protein Complex
(Sorting and Assembly Machinery)
(b-Barrell)
Figure 12-8 Molecular Biology of the Cell (© Garland Science 2008)
NuclearTransport
•Bidirectional•Single Large Pore Complex Spans 2 lipid bilayers•Nuclear Pores much larger than other translocons
Figure 12-9c Molecular Biology of the Cell (© Garland Science 2008)
EM of Transverse Section Showing a Side-View through two NPCs
Figure 12-9b Molecular Biology of the Cell (© Garland Science 2008)
Scanning EM of NPCs as Viewed from the Nucleoplasm
Figure 12-9a Molecular Biology of the Cell (© Garland Science 2008)
Structure of a Nuclear Pore Complex
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
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
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
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)
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
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
