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Biochemistry 4 Intracellular Compartments and Protein Sorting Mitochondria & Chloroplasts: organelles, transport to and from Dr Julia von Blume (MPI Biochemistry) Prof. Roland Beckmann Dr. Thomas Becker Literature Alberts, Bray, Hopkin, Johnson, Lewis, Raff, Roberts, Walter Essential Cell Biology 3rd edition, 2010, Garland Science Alberts, Johnson, Lewis, Raff, Roberts, Walter Molecular Biology of the Cell 5th edition, 2008, Garland Science Eurkaryotic cells are compartmentalized Features of cellular compartmentalization 1. cellular compartments are enclosed by a membrane 2. each compartment is endowed with a characteristic set of enzymes 3. each compartment contains specialized molecules 4. each compartment is equipped with specialized transport systems Eurkaryotic cells are compartmentalized protein synthesis and degradation ATP generation host of the DNA gene expression regulation degradation of cellular material protein synthesis lipid production Ca2+ store protein sorting lipid sorting distribution of proteins protein modification protein sorting The synthesis of all proteins starts in the cytosol Room1 Room II Room III How are proteins within cells targeted to their specific location within the cell? How are proteins within a cell targeted to a specific compartment? Proteins have intrinsic signals that govern their transport and localization in the cell Protein signal sequence (ss) Günther Blobel, Nobel Prize for Physiology or Medicine, 1999 Proteins are targeted to specific compartments by signal sequences - proteins are guided to correct cellular compartment by a signal sequence - recognition follows the biophysical properties of the sequence N-terminal (cleaved or uncleaved) C-terminal (uncleaved) internal (uncleaved) (positively charged amino acids) (hydrophobic amino acids) (hydroxylated amino acids) (negatively charged amino acids) resides in the cytosol. After the cDNA encoding this protein is transfected into cells, the location of the fusion protein is determined by immunostaining or by cell fractionation. in the in vitro Three method translocated in Approach to define signal sequences signal sequence a or b gene encoding cytosolic protein 1. The labeled with the organ plasmid used to transfect cells A A B signal sequence a ( ) directs fusion protein to organelle A B signal sequence b ( ) directs fusion protein to organelle B By altering the signal sequence using site-directed mutagenesis, we can determine which structural 3. The protein digestion whe added to the but is suscepti first added to organelle mem How does protein transport in eukaryotic cells work? There are three major protein transport principles for entry/transport between different compartments 1. gated transport 2. transmembrane transport 3. vesicular transport In gated transport proteins move between cytosol and nuclear pore complex - proteins move between cytosol and nucleus (topologically equivalent) - proteins move through the nuclear pore complex nuclear pore complex acts as gate that actively transports: - specific macromolecules - macromolecular assemblies nuclear pore complex also allows diffusion of smaller molecules. In transmembrane transport proteins are actively translocated across the membrane - transmembrane protein translocators move proteins across a membrane from the cytosol - protein must unfold to snake through the tanslocator transport from cytosol to the: endoplasmic reticulum lumen mitochondria chloroplasts in plant cells Wickner and Schekman, Science, 2005 In vesicular transport membrane enclosed vesicles transport proteins between different compartments - membrane enclosed transport intermediates (transport vesicles) ferry proteins from one compartment to the other - vesicles become loaded with cargo molecules derived from the lumen of the compartment as they bud and pinch off from its membrane - cargo becomes discharged into a second compartment by fusing with the membrane enclosing that compartment Function of mitochondria and chloroplasts photosynthesis electron transport oxidative phosphorylation Mitochondria and chloroplasts are derived from Gram-negative bacteria • Prokaryotic cells have no internal membranes • Plasma membrane provides all membrane-dependent functions: Pumping of ions, ATP synthesis, protein secretion, lipid synthesis Mitochondria and chloroplasts are derived from Gram-negative bacteria outer membrane: - beta-barrel proteins - mildly hydrophobic peptidoglycan inner membrane: - alpha-helical proteins - highly hydrophobic Mitochondria and chloroplasts are endosymbiotic organelles • eukaryotic cells started out as anaerobic organisms without mitochondria/chloroplasts • established a stable endosymbiotic relation with a bacterium whose oxidative phosphorylation system they subverted for their own use Hypothesis of the evolutionary origins of mitochondria and chloroplasts prokaryotic Accumulating biochemical data suggest that - the double membrane enclosing chloroplasts and mitochondria derived from the ancestral bacteria - the phagosomal membrane disappeared during organelle evolution Structure and function of mitochondria - double membrane enclosed organelles - specialized on ATP-Synthesis using energy derived from electron transport and oxidative phosphorylation two subcompartments: matrix space and intermembrane space membranes: outer and inner membrane - contain DNA and ribosomes + other components required for protein synthesis - most of the mitochondrial proteins are encoded in the cell nucleus - most proteins have to be imported from the cytosol into the mitochondria Structure and function of chloroplasts - double membrane enclosed organelles - specialized on ATP-Synthesis derived from photosynthesis three subcompartments: intermembrane space, stroma, thylakoid space membranes: outer, inner and thylakoid membrane (contains photosynthetic pigments) - contain DNA and ribosomes + other components required for protein synthesis - most of the chloroplast proteins are encoded in the cell nucleus Mitochondria and chloroplasts have transferred most of their genome into the nucleus most of the genes encoding mitochondrial or chloroplast proteins are encoded by nuclear genes requirement for import machineries mitochondrial genome encodes: - a few inner membrane proteins involved in electron transport reactions - ribosomal RNAs - some tRNAs How does the protein transport into mitochondria work? Proteins destined to matrix: N-terminal signal sequence Cleaved by signal peptidase Proteins destined to outer, many intermembrane and inner membrane proteins: internal signal sequence Signal sequence: not cleaved How are the proteins encoded by nuclear genes transported to mitochondria/ chloroplasts? Translocation into mitochondria depends on signal sequences and protein translocators complexes contain proteins that act as a) receptors for mitochondrial precursor proteins b) proteins that form translocation channels Signal sequences of mitochondrial proteins are necessary and sufficient for import and localization N-terminal signal sequence for import into mitochondria hydrophobic and and positively charged amino acids fold into an amphiphilic alpha helix front side hydrophilic back side hydrophobic binds into hydrophobic groove of the TOM20 receptor translocation Approach to test the significance of a signal sequence for mitochondrial import isolated mitochondria signal sequence is cleaved off TOM and SAM complex translocate mitochondrial proteins into the outer mitochondrial membrane TOM complex: (translocase of the outer mitochondrial membrane) - import of all nucleus-encoded mitochondrial proteins - transfers proteins across the outer membrane - helps with the insertion of transmembrane proteins in outer membrane ß-barrel proteins are passed to the SAM complex that helps with folding The TIM23 complex transports soluble proteins into the matrix and helps to insert proteins into the inner membrane TIM23 complex: (translocase of the inner mitochondrial membrane) transfers soluble proteins into the matrix helps with the insertion of transmembrane proteins in inner membrane complex spans two membranes one of the core components of the complex contains a hydrophobic alpha-helical extension that is inserted in the outer mitochondrial membrane complex is associated to mitochondrial Hsp70 on the matrix side, which acts as an import ATPase (ATPase hydrolyzes ATP to pull proteins through the pore) The TIM22 complex mediates the insertion of a subclass of inner membrane proteins TIM22 complex: INNER MITOCHONDRIAL MEMBRANE (translocase of the inner mitochondrial membrane) helps with the insertion of a subclass of inner membrane proteins inculding: transporters that move ATP/ADP and Phosphate in and out of the mitochondria MATRIX The OXA complex mediates the insertion of inner membrane proteins synthesized by mitochondria OXA complex: mediates insertion of inner membrane proteins that are synthesized within the mitochondria helps to insert some imported inner membrane proteins that are initially transported to into the matrix space by other complexes Mitochondrial protein import Which energy sources drive the directional transport of the precursor proteins across the mitochondrial membranes? two different energy sources: ATP hydrolysis membrane potential across the inner mitochondrial membrane ATP hydrolysis and a membrane potential drive protein import into the mitochondria Import to the mitochondria requires ATP hydrolysis: a) outside the mitochondria release of the unfolded protein from Hsp70 proteins requires ATP hydrolysis b) import into the matrix by TIM23 mitochondrial Hsp70 pulls the precursor protein through the translocation channel release of the unfolded protein from mitochondrial Hsp70 requires ATP ATP hydrolysis and a membrane potential drive protein import into the mitochondria translocation of the transported precursor protein through the TIM channel requires membrane potential (H+gradient across the inner mitochondrial membrane) the signal sequence of the precursor protein is translocated into the matrix space in a process that requires a membrane potential across the inner membrane ATP hydrolysis and a membrane potential drive protein import into the mitochondria How does mitochondrial hsp70 drive import? Two models: trapped in matrix back and forth sliding active pulling of hsp70 Gram negative bacteria and mitochondria insert their beta barrel proteins in a similar way One of the central units of the SAM complex is homologous to the bacterial outer membrane protein that helps to insert beta-barrel proteins into the bacterial outer membrane from the periplasmic space conserved pathway if inserting of beta barrel proteins is further evidence for the endosymbiotic theory How are proteins inserted into the intermembrane space and inner membrane of mitochondria? Transport into the inner mitochondrial membrane and intermembrane space occurs via similar routes Insertion of proteins into the inner membrane: TOM TIM23 Transport into the inner mitochondrial membrane and intermembrane space occurs via similar routes Insertion of proteins into the inner membrane: Transport into the inner mitochondrial membrane and intermembrane space occurs via similar routes Insertion of proteins into intermembrane space: Transport into the inner mitochondrial membrane and intermembrane space occurs via similar routes Insertion of multispan membrane proteins: Mitochondria are the sites of ATP synthesis: proteins + small metabolites required outer membrane is permeable, inner membrane is impermeable metabolite-specific transporters required for transport of small molecules across the inner membrane transporters of inner membrane are multipass membrane proteins without cleavable signal sequence How does the protein transport into chloroplasts work? Thylakoids are membrane enclosed compartments Photosynthetic system ATP synthase Two signal sequences direct proteins to the thylakoid membrane in chloroplasts precursor proteins have a hydrophobic thylakoid signal sequence followed the N-terminal chloroplast signal sequence 1. proteins pass across the double membrane into the stroma across outer membrane: TOC across inner membrane: TIC 2. translocation to the thylakoid membrane or into thylakoid space There are four routes of protein translocation across/into the thylakoid membrane 1. Sec pathway: uses components that are homolog to the Sec proteins in bacteria which mediate the translocation across the bacterial plasma membrane 2. SRP-like pathway: uses the homolog of the signal recognition particle, or SRP 3. TAT pathway: twin arginine translocation, requires H+ for translocation 4. spontaneous insertion pathway: does not need any protein translocation Protein transport into chloroplasts mostly resembles transport into mitochondria Similarities between protein transport to mitochondria and chloroplasts 1. import of most proteins occurs post translationally 2. use separate translocation complexes in each membrane 3. energy requirement 4. use of amphiphilic N-terminal signal sequences that are removed after use Differences in mitochondrial and chloroplast protein transport Differences in mitochondrial and chloroplast protein transport 1. subcompartments mitochondria, 2; chloroplasts 3 2. protein components for import differ 3. chloroplasts use GTP- and ATP hydrolysis to power the transport of proteins across their inner membrane whereas mitochondria use membrane potential 4. chloroplasts use H+gradient for transport across the thylakoid membrane 5. signal sequences are different because plant cells have chloroplasts and mitochondria Take home message • although mitochondria and chloroplasts have their own genetic systems they produce only a small proportion of their own proteins • chloroplasts and mitochondria import most of their proteins from the cytosol using similar import mechanisms • proteins are transported in an unfolded state across outer and inner membrane simultaneously into the matrix space or stroma • Both ATP hydrolysis and membrane potential across the inner membrane drive translocation into mitochondria, whereas GTP and ATP hydrolysis drive translocation into chloroplast • Chaperones of the Hsp70 family keep the protein in a unfolded state in the cytosol or mitochondrial Hsp70 chaperones pull the polypeptide chain into the matrix or stroma • Translocation requires a signal sequence (N-terminal, internal) • Import into the thylakoid membrane can occur by several routes depending on the chaperones and energy source used.