This preview shows page 1. Sign up to view the full content.
Unformatted text preview: Eukaryotic cells are highly compartmentalized Each compartment contains a distinct set of proteins in its membrane and within its interior.
Vertebrate cells have ~ 10 billion proteins, many of which need to be sorted Assigned Reading for next class: Chapter 12 (pg. 704 -712) Most proteins are made in the cytosol, yet need to be sorted/targeted to their specific site of function to do their job Map of intracellular transport routes ribosomes cytoplasm Three distinct types of routes Targeting to an intracellular destination requires a specialized amino acid sequence or sorting signal in the protein Signal sequences on proteins function as zip-codes that specify their fast delivery to a specific neighborhood Signal sequences are necessary and sufficient to specify delivery to a location, They are also referred to as localization sequences, import signals, etc Note that most signal sequences have a hydrophilic character, but those specifying delivery to a membrane, have a hydrophobic character The case of mitochondrial biogenesis:
Exclusive to the inner membrane , because outer membrane has large -diameter porins Mitochondria in yeast: a dynamic tubular compartment ~500-600 proteins are imported; ~ 10% of the yeast proteome Approaches to study signal sequences and protein translocation across membranes: Mitochondrial targeting information is contained within a signal sequenceClassic analysis of signal sequences: necessary versus sufficient Biochemical properties of signal sequences (presequences)- signal sequences are amphiphilic
And a receptor for signal sequences in the outer mitochondrial membrane… Unassisted translocation…? No way! The search for a TRANSLOCON using reconstituted systems… Reconstituted protein import allowed the dissection of the mechanisms of protein translocation and subsequent identification of the translocon
Cell-free translation Radiolabeled ‘unfolded’ precursor Cell extract [ribosomes, tRNA’s, etc.] Radiolabeled amino acids [S35] mRNA Protease Sensitive (control) Translocation intermediate Isolated mitochondria Protease resistant Proteins transiently span the inner and outer mitochondrial membranes during their translocation; such transient intermediates can be captured at low temperature Evidence that mitochondrial precursors must be unfolded to be competent for translocationDihydrofolate reductase (DHFR) folds into a ‘rock solid’ globule upon binding its ligand methotraxate (MTX). It even resists urea denaturation. The chemical urea (at a 6-8 molar concentration) can mimic the unfolding effect of cytosolic chaperones Translocation occurs at contact sites Use of chemical crosslinkers to identify components of the translocon- BPTI (pancreatic bovine trypsin inhibitor) folds tightly due to several intramolecular disulfide bonds Functionally similar to DHFR for the purpose of these experiments Morphology of purified TOM complexes by cryo EM- Pore diameter = 2 nm Why three pores together? Field of view Individual complexes Computer-assisted image averaging 10 This can now be explained… Four mitochondrial transloconsTranslocons are ligand activated channels The ligand is the signal sequence Used for all nucleus -encoded proteins Used for multipass beta barrel proteins Different routes for mitochondrial protein import using different translocons Connects TOM with TIM Used for multipass proteins …and the tiny TIMs Used for proteins encoded by mitochondrial genome, and for some inner membrane proteins Cytosolic ‘chaperones’ maintain the precursors unfolded prior to translocation- Targeting of mitochondrial protein precursor to the TOM complex: Binding relay model for its targeting and initial translocation across membranes
Delivery of the substrate to the mitochondrial membrane is aided by receptors that capture the signal sequence and transfer it from one receptor to another until it reaches the TOM translocon An affinity gradient imparts directionality? Sequence of events in mitochondrial protein import The thermal-ratchet model for protein translocation across the inner membrane Stochastic sliding within the translocon, and sequential trapping in the matrix Hsp70’s are a family of cellular proteins that bind the unfolded state of polypeptides ‘Proposed hand-over-hand’ binding of multiple Hsp70p proteins The cross-bridge ratchet model for translocation across the inner membrane- Hsp70p proteins pull on the translocating chain, while anchored to the TIM translocon Many translocons have a “J domain” by the exit tunnel. J domains bind Hsp70s and accelarate their ATP hydrolysis rate Protein translocation into the mitochondrial matrix requires energy at three steps: 1. Dissociation of chaperones 2. Electrophoresis pulls the positively-charged sequence 3. Release of chaperones Energy-requiring steps during protein translocation into mitochondria: 1. 2. The release of cytosolic Hsp70’s from mitochondrial precursor is ATP-hydrolysis dependent; it can be bypassed by adding the precursor in an unfolded state. The membrane potential across the inner mitochondrial membrane and an electrochemical proton gradient ‘pull’ the signal through the TIM translocon by electrophoresis. ATP hydrolysis by mitochondrial Hsp70’s pulls and releases the translocated protein. 3. Summary of protein translocation into the mitochondrial matrix and protein folding- Nascent polypeptide chains are produced in ribosome's and are kept in “translocation -competent state” by chaperones, or by pausing translation. A “signal sequence” in the nascent chain specifies intended organelle target. The nascent chain is targeted to a “translocon” directly, or via a receptor. The polypeptide is translocated (or inserted in the membrane) in a reaction that involves energy from any of several sources: ATP hydrolysis & membrane potential. The nascent chain finally folds, unassisted, or assisted by chaperones. Mechanisms for protein insertion into the outer mitochondrial membraneIntegration of a multi-pass porin into the membrane via the TOM and SAM complexes… (also mask hydrophobic AA side chains) The outer mitochondrial membrane is full of porins, so it is permeable to inorganic ions and metabolites, but not to proteins Mechanisms for insertion of single pass proteins into the inner mitochondrial membrane- The most common route… A hydrophobic sequence next to the signal sequence acts as a stop-transfer signal at TIM, without affecting translocation through TOM Stop-transfer pathway A second route… Cleavage of the signal sequence in the matrix exposes a second signal sequence that targets the precursor to the OXA translocon, which mostly translocates endogenous proteins Conservative pathway
Endogenous pathway Mechanism of protein targeting to the inter-membrane spaceAfter its insertion into the inner mitochondrial membrane via the TOM complex, a signal peptidase in the inter-membrane space cuts off the membrane domain Mechanisms for insertion of multipass-proteins into the inner mitochondrial membraneThese contain an internal signal sequence , which is also a trans-membrane domain They are first inserted as loops trough the TOM translocon Chaperones in the inter-membrane space capture the precursor and deliver it to the TIM22 translocon, which specializes on this type of multipass proteins Protein translocation across the chloroplast membranes- Fig. 1. Conserved translocation themes. (A) Either chaperones (red) or occupancy of the C-terminal region of nascent chains in the ribosomal exit tunnel (blue) prevents premature stable folding of pre -proteins. Membrane-bound receptors (green) bind preproteins and transfer them into the translocon (purple), which can conduct them across the bilayer or release apolar regions laterally into the bilayer. Motors and chaperones (yellow) on the trans surface of the membrane complete the transport task. (B) Cotranslational translocation into the ER. (C) Mitochondrial protein import [adapted from Wiedemann et al. (94)]. OM, outer membrane, IMS, intermembrane space, IM, inner membrane. (D) Chloroplast protein import Diversity of bacterial translocation pathwaysThe bacterial translocon of the inner membrane (SecY) is evolutionarily related to the translocon in the eukaryotic ER membrane (Sec61); thus can study bacteria to understand eukaryotes ...
View Full Document
- Fall '10