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BIOL340 Molecular Cell Biology Week 4 Protein Sorting Mechanisms Dr Jody Gorman [email protected] Becker’s World of the Cell. 9th Edition. Chpts 12, 16 and 19 Molecular Biology of the Cell, 7th ed. Various chapters 1 Overview Protein synthesis and folding Protein road map Sorting signals Protei...

BIOL340 Molecular Cell Biology Week 4 Protein Sorting Mechanisms Dr Jody Gorman [email protected] Becker’s World of the Cell. 9th Edition. Chpts 12, 16 and 19 Molecular Biology of the Cell, 7th ed. Various chapters 1 Overview Protein synthesis and folding Protein road map Sorting signals Protein trafficking to the nucleus, mitochondria, chloroplasts and peroxisomes Summary ROAD TRIP!!! But which way do we go? 2 Review of protein synthesis 3 Review of protein folding A protein must be converted from a linear chain of amino acids to a specific 3D shape (conformation) to gain its function. Unfolded Folded Cellulose " Function Endoglucanase el lul ose C 4 Proteins required in various cellular compartments Intracellular processes must be separated ¢semipermeable membranes But if most proteins begin their synthesis in the cytosol… How are they accurately targeted to their appropriate compartment? Figure 12.1. Molecular Biology of the Cell 5 So many possible destinations… Will remain in cytosol or be transported to nucleus, mitochondria, chloroplasts or peroxisome Will enter the ER/ER membrane or be transported out of cell or into endomembrane system organelles 6 Lodish et al. (2000) Protein Road Map Three distinct routes of protein importation into organelles ¢ Through nuclear pores ¢Across membranes ¢Via vesicles 7 Protein Road Map Importation can occur →co-translationally OR →post-translationally 8 Figure 12.24. Molecular Biology of the Cell Protein Road Map cytoplasm 1 1 Proteins released into cytosol from free ribosomes Proteins synthesised on ribosomes attached to ER (RER) nucleus mitochondria Vesicular transport plastid endoplasmic reticulum Gated transport Transmembrane transport peroxisome lysosome golgi secretory vesicles endosome cell surface 9 How does a protein know where to go? Its fate depends on sorting signals or “postcode” Info stored in the amino acid sequence Sorting signals can be built into a protein in two ways Signal peptide ¢ER Signal patch ¢Vesicular transport (e.g. Golgi to lysosome) 10 Sorting Signals Sorting Signals Signal sequences were first discovered in proteins imported into the rough ER Two reactions – one with rough microsomes from the ER → size difference = short N-terminal sequence Figure 12.18. Molecular Biology of the Cell 12 Sorting Signals Some typical signal sequences (aka signal peptides) Figure 12.13. Molecular Biology of the Cell 13 Protein Road Map cytoplasm nucleus peroxisome mitochondria plastid endoplasmic reticulum lysosome golgi secretory vesicles endosome cell surface 14 Transportation into the nucleus Figure 16.27. Becker’s World of the Cell 15 Transport of proteins between the nucleus and cytosol Continuous bidirectional transport across channels in the nuclear envelope - selective → Histones, DNA and RNA polymerases, gene regulatory proteins, and RNA processing proteins all require import from the cytosol where they are made → tRNA’s and mRNA’s synthesized n the nucleus are transported into the cytosol where they participate in translation 16 Transport of proteins between the nucleus and cytosol The lumen of the nuclear envelope is continuous with the ER lumen Bidirectional traffic occurs through the nuclear pore complexes Mr = 125,000,000 Da ~ 50 different proteins (nucleoporins) 17 Figure 12.54A. Molecular Biology of the Cell The Nuclear Pore Complex (NPC) Nuclear pore complexes perforate the nuclear envelope Figure 12.55. Molecular Biology of the Cell Freely permeable to small water soluble molecules (< 5000 Da) Molecules > 60,000 Da cannot pass Proteins with a nuclear localization signal (NLS) are recognised by nuclear import receptors RNA and new ribosomal subunits have nuclear export signals recognised by nuclear export receptors 18 Nuclear Localisation Signals (NLS) Nuclear localisation signals direct proteins to the nucleus Figure 12.56. Molecular Biology of the Cell 19 Nuclear Import Receptors Most nuclear localisation signals are specifically recognised by nuclear import receptors These are soluble cytosolic proteins that bind to both the NLS on the protein being transported and to NPC proteins Figure 12.54A. Molecular Biology of the Cell 20 Transport through the Nuclear Pore Complex Interaction between FG repeats on fibrils/filaments and binding sites on nuclear import receptors enables transport across the nuclear pore complex 21 Figure 12.59. Molecular Biology of the Cell Nuclear transport The Ran GTPase Imposes Directionality on Nuclear Import Through NPCs The critical difference between Ran-mediated nuclear import and nuclear export is the nature of cargo binding by the cargo receptor. In nuclear import, cargo binding is mutually exclusive of Ran-GTP; in nuclear export, cargo binding requires RanGTP. 22 Figure 12.61. Molecular Biology of the Cell Protein Road Map cytoplasm nucleus peroxisome mitochondria plastid endoplasmic reticulum lysosome golgi secretory vesicles endosome cell surface 23 Mitochondria Membrane bound organelles convert energy to forms that drive the cell (ATP) Matrix - hundreds of enzymes (TCA cycle) Inner membrane - ATP synthase etc. Outer membrane - porin Intermembrane space - several kinases Figure 12.47A. Molecular Biology of the Cell 24 Importing proteins into mitochondria and chloroplasts Translocation Into Mitochondria Depends on Signal Sequences and Protein Translocators Mitochondrial proteins are imported post-translationally as unfolded polypeptide chains Protein import is powered by ATP hydrolysis, a membrane potential, and redox potential Transport into the inner mitochondrial membrane occurs via several routes 25 Importing proteins into mitochondria Multi-subunit protein complexes that function as protein translocators mediate movement across mitochondrial membranes Figure 12.48A. Molecular Biology of the Cell Importing proteins into mitochondria TOM = Translocator, Outer Membrane TIM = Translocator, Inner Membrane ATP hydrolysis drives dissociation of protein from cytosolic Hsp70 and matrix mtHsp70 27 Figure 12.49. Molecular Biology of the Cell Importing proteins into the inner mitochondrial membrane Combination of translocator protein complexes, signal sequences and sometimes chaperones mediates movement into the mitochondrial membrane 28 Figure 12.51. Molecular Biology of the Cell 29 Chloroplasts Specialized plastid (photosynthesis) 6 compartments inner and outer envelope membrane intervening membrane space stroma thylakoid membrane lumen Figure 12.47B. Molecular Biology of the Cell 29 Importing proteins into chloroplasts Translocation into chloroplasts similar to mitochondrial transport → Occurs post-translationally → Uses separate translocator complexes in each membrane (TOC and TIC) → requires energy → Uses N-terminal signal sequences that are ckeaved after use TOC = Translocator, Outer Membrane of Chloroplasts TIC = Translocator, Inner Membrane of Chloroplasts 30 Chloroplast protein import mechanisms are similar to those of mitochondria Two signal sequences required for transport to the thylakoid lumen 31 Figure 12.53A. Molecular Biology of the Cell Chloroplast protein import mechanisms are similar to those of mitochondria There are several pathways that transport proteins from the stroma to the thylakoid space Hydrolysis of ATP and GTP drives import 32 Figure 12.53B. Molecular Biology of the Cell Protein Road Map cytoplasm nucleus peroxisome mitochondria plastid endoplasmic reticulum lysosome golgi secretory vesicles endosome cell surface 33 34 Peroxisomes electron micrograph of three peroxisomes in a rat liver cell Single membrane organelle Oxidizes organic molecules Use O2 to remove H2 Produces and degrades H2O2 Breakdown of fatty acids ¢ (Acetyl coA -> TCA cycle) Ancient metabolic organelle? Figure 12.43. Molecular Biology of the Cell 34 Importing proteins into peroxisomes Surrounded by only a single membrane Acquire most proteins by selective import from the cytosol but some enter the via the ER Short signal sequences direct the import of proteins into peroxisomes Soluble Peroxisomal Targeting Signal (PTS) receptors and docking receptors on the cytosolic surface help Driven by ATP hydrolysis 35 Import into Peroxisomes Ma, Changle & Agrawal, Gaurav & Subramani, Suresh. (2011). Peroxisome assembly: Matrix and membrane protein biogenesis. The Journal of cell biology. 193. 7-16. Mechanisms not well understood >23 proteins involved in the import process - peroxins A complex of at least 6 different proteins forms a translocator Imported in native confirmation aided by at least one soluble import receptor, peroxin 5, Pex5 Pex5 carries its cargo all the way into the peroxisome before being recycled 36 Summary Although most proteins are synthesised in the cytoplasm, they are directed to their final compartmental location by specific sorting signals Proteins synthesized in the cytosol may traffic to either the nucleus, mitochondria, chloroplast or peroxisome Nucleus: bidirectional transport through nuclear pore complex (NLS + importins/exportins, Ran-GTP + pore complex) Mitochondria: signal sequence + chaperones + TOM/TIM complex + ATP Chloroplast: similar to mitochondria (signal sequences, chaperones, TOC/TIC, ATP/GTP) Peroxisomes: PTS/mPTS + PTS receptors 37 Example Exam Questions Describe the experiments that could be carried out to confirm that a sorting signal is directing a protein to the nucleus (3 marks) Describe, with the aid of a diagram, how cytosolic proteins are trafficked to the mitochondrial matrix (10 marks) Identify and briefly describe the two ways sorting signals can be built into a protein (3 marks) Describe, with the aid of a diagram, how cytosolic proteins are trafficked to the nucleus (10 marks) 38