Protein Sorting Lecture 13 PDF

Protein Sorting I Nucleus, Mitochondria, and Peroxisomes ECB6 Chapter 15 pp. 516-526 MBOC7 Chapter 12 pp. 723-745 So many membrane-bound compartments in a eukaryotic cell! Nucleus Mitochondria...

Protein Sorting I Nucleus, Mitochondria, and Peroxisomes ECB6 Chapter 15 pp. 516-526 MBOC7 Chapter 12 pp. 723-745 So many membrane-bound compartments in a eukaryotic cell! Nucleus Mitochondria Chloroplasts (plant) Endomembrane system nuclear envelope, ER, golgi, endosomes, lysosomes, vesicles Peroxisomes  Some of these organelles we can see in this slice 5 1 from a mouse liver cell 2 3 4 Organelles occupy about half the volume of a cell How do proteins get delivered to the right address?  Organelles import proteins by one of three mechanisms  Visualizing “traffic patterns” in a eukaryotic cell CHLOROPLAST Where is transport unidirectional? Where is transport bidirectional? PLASMA MEMBRANE and CELL EXTERIOR The synthesis of all nuclear-encoded proteins starts in the cytoplasm Signal sequences that are part of the amino acid sequence of a protein are used to direct import into an organelle. The import signal is like a “postal code” Signal sequences direct proteins to the right location Features N-terminus Rich in hydrophobic aa C-terminus KDEL N-terminus Amphipathic helix with basic aa on one face Anywhere Rich in basic aa Anywhere Alternating hydrophobic aa Often at C-terminus SKL What do you think happens to proteins that lack a signal sequence? A.Degraded B.Enters the nucleus C. Sent to the ER D. Stays in the cytosol Gated Transport  Nuclear pores are the sole gateway between the nucleus and the cytoplasm Proteins enter and leave the nucleus in their fully folded state through nuclear pores Some proteins enter and exit multiple times Two ways to pass through a nuclear pore complex depending on size Hydrogel pore interior Small proteins “size exclusion chromatography” ≤ 40 kDa Diffusion Large proteins Protein complexes Viruses ≥ 40 kDa Active transport Nuclear import receptors bind to cargo an appropriate NLS and subunits of the nuclear pore NLS = nuclear localization signal is an encoded part of the protein Families of receptors recognize a wide variety of different NLS sequences on proteins The function of a nuclear localization signal Nuclei Cytoplasm The compartmentalization of Ran-GDP and Ran-GTP provides directionality Energy supplied by GTP hydrolysis Nuclear import cycle drives nuclear transport The structure of a nuclear import receptor shows how Ran-GTP binding causes the release of cargo The transport receptor has an S-shape Ran-GTP binding on the nuclear side disrupts interaction with NLS on cargo Ran-GTP binding pushes on this Cargo is released loop and expels the cargo  Nuclear export works like nuclear import, but in reverse Nuclear import (NLS) Stretch of 5-6 basic residues at C-terminus (e.g. KKKKRK) Nuclear export (NES) Short stretch of alternating hydrophobic residues  NFᴋB regulates our immune response to infection. Under normal conditions, IᴋB binds to NFᴋB blocking the NLS of NFᴋB Infection so NFᴋB remains inactive in the cytosol. Normal Upon infection, IκB is ubiquitinated and degraded, releasing NFᴋB. The nuclear localization signal of NFᴋB is unmasked leading to its nuclear import. Nuclear NFᴋB turns on genes that fight infection. Eventually, NFᴋB is transported back to the cytosol to await the next cellular stress signal. Which of the following would lead to decreased nuclear localization of NFᴋB after interleukin stimulation? Choose one or more. a.A large amount of short peptide containing an NLS is added to the cells. b.The ubiquitination sites on IᴋB are altered so ubiquitination no longer occurs. c.Ran is bound to a nonhydrolyzable analog of GTP. d.Ran-GEF is kept in an active form in the nucleus. Three modes of protein transport Protein Translocation Mitochondria Chloroplasts* May have evolved from proteins already present in the host before endosymbiosis  From endosymbiont to organelle 1. An endosymbiont must be able to reproduce inside its host 2. Over time, genes from the endosymbiont are transferred to the host nucleus 3. Evolution of translocators and signal sequences to allow the import of nuclear- encoded proteins 4. Genes essential for endosymbiont reproduction are eventually lost from the endosymbiont and provided by host 5. The endosymbiont genome is gradually reduced to bare essentials The signal sequence for Mt import is an amphipathic - helix usually found at the N-terminus of a polypeptide Charged basic residues on one side of helix Hydrophobic residues other e.g. cytochrome side of helix oxidase Binding pocket on receptor proteins, part of translocation channel in OMM  Mitochondrial precursor proteins are unfolded during import Which end of the protein enters first? Watch Movie 15.2 Why signal cleavage? TOM and TIM23 complexes can link up to form a continuous translocation channel across the Mt double membrane TOM = Translocator of the OUTER Membrane Receptors in TOM complex bind to the signal helix Diffusion brings the two translocators together at contact sites so a polypeptide can be threaded across both membranes at once into the matrix TIM= Translocator of the INNER Membrane Protein import into the mitochondria requires energy (Active import) What would happen in each of the following cases? Assume in each case that the protein involved is soluble, not a membrane protein. A. You add a signal sequence (for the mitochondria) to the amino-terminal end of a normally cytosolic protein. B. You change the charged amino acids in a mitochondrial signal sequence into acidic amino acids. C. You change the hydrophobic amino acids in a mitochondrial signal sequence into other, hydrophobic, amino acids. D. You move the amino-terminal mitochondrial signal sequence to the carboxyl-terminal end of the protein.  What about proteins that reside in other spaces in mitochondria? TIM22 puts multi-pass SAM complex inserts transmembrane into the IMM porins into the OMM  Peroxisomes ENDOPLASMIC RETICULUM Organelles that use oxygen and produce hydrogen peroxide Bud off from the ER, divide by fission Receive membrane and associated proteins from the ER Receive matrix proteins from the cytosol Peroxisomes carry out oxidation reactions Reactions use O2 as an electron acceptor thereby generating H2O2 which is recycled into H20 and O2 by catalase Involved in the production of cholesterol and specialized lipids of the myelin sheath (heart, brain) Site of breakdown of lipid-soluble organic molecules (e.g. ethanol, long-chain fatty acids) and conversion of uric acid and amino acids into soluble forms for excretion. Rich in enzymes (e.g. catalase and urate oxidase) which crystalize during EM prep resulting in an electron-dense cores.  Soluble protein import into peroxisomes combines features of mitochondrial and nuclear import Proteins are imported in a folded state through a translocation channel An import receptor (R) that recognizes C-terminal SKL peroxisome import signals on soluble proteins in the cytosol ATP R R Ub The receptor shuttles cargo into the peroxisome and unloads it. The SKL signal sequence on the cargo is not cleaved off. The receptor is ubiquitinated allowing its return to the cytosol where it picks up new cargo. Export of the receptor back to the cytosol Which features resemble nuclear transport? requires ATP hydrolysis by a complex in the Which features are resemble Mt import? peroxisome membrane Catalase, an enzyme normally found in peroxisomes, is present in normal amounts in cells that do not contain peroxisomes. It is possible to determine the location of catalase in such cells, using immunofluorescence microscopy with antibodies specific for catalase. Fluorescent micrographs of normal cells and two peroxisome deficient-cell lines are shown in the figure below. Where is catalase likely to be located in cells without peroxisomes? Why does catalase show up as small dots of fluorescence in normal cells? Normal cell Mutant 1 Mutant 2  Protein Sorting II Co-translational import into the ER Chapter 15 ECB6 pp. 526-532 Chapter 12 MBoC7 pp. 698-723