Transcript Powerpoint file

Thursday 9/4 2014
Mike Mueckler
[email protected]
Intracellular Targeting of Nascent Polypeptides
Mitochondria are the Sites of
Oxidative ATP Production
Sugars
Figure 14-10 Molecular Biology of the Cell (© Garland Science 2008)
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
Proteins are Incorporated Into Mitochondria
Via Several Different Routes
Figure 12-23 Molecular Biology of the Cell (© Garland Science 2008)
Targeting to the Matrix Requires an NTerminal Import Sequence
N-terminal Import Sequences Form Amphipathic a
Helices that Interact with the Tom20/22 Receptor
Hydrophobic cleft
Figure 12-22 Molecular Biology of the Cell (© Garland Science 2008)
Protein Import into the Matrix
Requires Passage Through
Two Separate Membrane Translocons
Proteins Traverse the TOM and TIM
Translocons in an Unfolded State
Translocation into
the Matrix Occurs at
Zones of Adhesion
Protein Import into the Matrix Requires ATP
Hydrolysis and an Intact Proton Gradient
Across the Inner Membrane
Figure 12-26 Molecular Biology of the Cell (© Garland Science 2008)
Targeting to the Inner Membrane Occurs Via 3 Distinct Routes
Stop-Transfer-Mediated
Single-Pass
Proteins
Oxa1-Mediated
Tom70/Tim22/54-Mediated
Multi-Pass Proteins
ADP/ATP Antiporter
Cytochrome oxidase
subunit CoxVa
ATP Synthase Subunit 9
Targeting to the Intermembranous Space
Occurs Via Two Distinct Pathways
IM Space Protease
Cytochrome B2
Direct Delivery
Cytochrome c Heme Lyase
Targeting to the Outer Membrane
Via the SAM Protein Complex
(Sorting and
Assembly
Machinery)
(b-Barrell)
Figure 12-27 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
Figure 12-8 Molecular Biology of the Cell (© Garland Science 2008)
EM of Transverse Section Showing a
Side-View through two NPCs
Figure 12-9c Molecular Biology of the Cell (© Garland Science 2008)
Scanning EM of NPCs as Viewed from
the Nucleoplasm
Figure 12-9b Molecular Biology of the Cell (© Garland Science 2008)
Structure of a Nuclear Pore Complex
Figure 12-9a Molecular Biology of the Cell (© Garland Science 2008)
Gated Diffusion Barrier Model of
Nuclear Transport
Meshwork of disordered
protein domains
containing FG repeats
Figure 12-10 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 12-11 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 12-12 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 12-13 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 12-14 Molecular Biology of the Cell (© Garland Science 2008)
Nuclear Import and Export Operate Via Reciprocal
Use of the Ran-GDP/GTP Concentration Gradient
Figure 12-15 Molecular Biology of the Cell (© Garland Science 2008)