TARGETING OF NASCENT POLYPEPTIDES OUTSIDE OF THE SECRETORY PATHWAY

Thursday 9/4 2014Mike Mueckler

mmueckler@wustl.edu

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