Lecture 11 Protein Sorting I 2023.pdf

Protein Sorting: Nucleus and Mitochondrion NPCs from inside of nucleus 1 Protein Sorting • “Sorting”: movement to appropriate destinations (compartments). • Eukaryotic cells are compartmentalized. • Each organelle requires specific proteins (e.g. enzymes, transporters etc.) to perform their func...

Protein Sorting: Nucleus and Mitochondrion NPCs from inside of nucleus 1 Protein Sorting • “Sorting”: movement to appropriate destinations (compartments). • Eukaryotic cells are compartmentalized. • Each organelle requires specific proteins (e.g. enzymes, transporters etc.) to perform their function. • About 10 billion proteins of 10 – 20 thousand types. • Most of these are synthesized elsewhere in the cytosol and must be transported (“sorted”) to appropriate sites. 2 Movement of Proteins Between Compartments 1. Gated transport: nucleus 2. Transmembrane transport: mitochondrion and ER 3. Vesicular transport: secretory pathway 3 Signal Sequence or Patch • Amino acid sequence that directs protein to specific destination. • Signal sequence: signal is composed of consecutive amino acid sequence at terminal end of protein. • Signal patch: signal amino acids are internal to protein. 4 Protein Sorting Between Cytosol and Nucleus • Important proteins, such as polymerases, gene regulatory proteins etc. are synthesized in the cytosol and imported into the nucleus. • Others (e.g. mRNA) are synthesized in the nucleus and exported to the cytosol. • Bidirectional traffic • Nuclear envelope composed of inner and outer membrane, and nuclear pore complexes. 5 Nuclear Pore Complex • • • • Perforate nuclear envelope. Octagonal structure. Composed of nucleoporins: ring, scaffold and channel. Allow passage of molecules. 6 Nuclear Pore Complex • Structure includes an aqueous pore. • Small proteins traverse by passive diffusion. • Recent evidence indicates a tangled meshwork lines the pore to block passive diffusion of large molecules. • Large proteins traverse by active transport mechanism. • Proteins are imported/exported in folded conformation. 7 Figure 12-10 Molecular Biology of the Cell (2008) Nuclear Import Receptors • • • • • • Bind to nucleoporins and nuclear localization signals (NLSs). NLSs are signal sequences or patches. NLS-receptor recognition initiates import of cargo proteins. NLSs are specific for receptors. Sometimes adaptor proteins involved. Export works the same way, but in reverse, and utilize export receptors. NLS indicated by red circles 8 Directional Transport • Import and export consumes energy. • A GTPase called Ran acts as a molecular switch. • Directionality occurs primarily because Ran-GDP is concentrated in the cytosol, while Ran-GTP exists in the nucleus. • This gradient of two conformational forms of Ran drives nuclear transport in the appropriate direction. 9 Nuclear Import Nuclear Export (from receptors) (to receptors) 10 Compartmentalization of Ran-GDP and Ran-GTP • • • • Ran is a molecular switch that exists in two conformations: Ran-GDP in cytosol, Ran-GTP in nucleus. GTPase-activating protein (GAP) hydrolyzes GTP, converting RanGTP to Ran-GDP. Guanine exchange factor (GEF) promotes GDP-GTP exchange, converting Ran-GDP to Ran-GTP. The activity of GAP and GEF maintain the Ran-GDP/Ran-GTP gradient that drives import and export. Fig. 12-15, Alberts 4th ed. 11 Protein Sorting to the Mitochondrion • Many mitochondrial proteins are necessary for electron transport, oxidative phosphorylation, and ATP synthesis. • Most proteins must be encoded in the nucleus and imported from the cytosol. • Posttranslational translocation. • Double membrane structure. • Proteins are transported to matrix or inserted into membrane. Fig. 12-22 Alberts 12 The Mitochondrial Signal Sequence • Exposed amino acid sequence that directs protein to appropriate address. • Mitochondrial signal sequence is modified as an a helix. • (+) charged amino acids are exposed at one side, uncharged ones at the other. 13 Protein Translocator Complexes 1. 2. 3. TOM: translocase of the outer mitochondrial membrane. TIM: translocase of the inner mitochondrial membrane. OXA complex 14 Protein Translocator Complexes 1. 2. 3. TOM: imports all mito-destined proteins to intermembrane space; membrane insertion at outer membrane. TIM: imports proteins to matrix (TIM 23); insertion into inner membrane (TIM 23); insertion of carrier proteins (TIM 22). OXA complex: insertion of proteins into inner membrane. 15 Translocation Across the OM Formation of precursor protein 1 Interacting proteins (e.g. chaperones, cytosolic Hsp70) maintain unfolded structure 2 3 Binding and recognition of signal sequence at TOM receptor proteins (TOM 20, TOM 5) Transfer of signal sequence and polypeptide to TOM channel (TOM 40) 4 Interacting proteins (e.g. Hsp70) stripped away by ATP hydrolysis 5 Unfolded polypeptide fed through TOM channel 16 Energy released by ATP hydrolysis pushes polypeptide chain through TOM channel. 4 1 2 3 5 6 7 8 9 Note: The TOM complex is composed of multiple proteins, such as smaller receptor proteins (TOM 20 and TOM 5) and a pore-forming region (TOM 40). 17 Figure 12-26 Molecular Biology of the Cell (© Garland Science 2008) Translocation Across the IM 6 Polypeptide bound to TIM complex Electrochemical H+ gradient pulls (+) charged signal sequence through TIM 7 8 9 Signal sequence cleaved away by matrix processing peptidase (MPP), and mitochondrial Hsp70 binds ATP hydrolysis induces conformational change of mitochondrial Hsp70, polypeptide is pulled through 18 Cross-Bridge Ratchet Model • Mitochondrial Hsp70 associated with TIM 23. • ATP hydrolysis drives conformational change in Hsp70 that actively pulls chain through. 19 Insertion into the Inner Membrane SS reaches matrix Chain pulled into matrix 2nd SS binds to TIM 23 and stops translocation 1st SS cleaved 2nd SS guides chain to OXA Insertion in IM by OXA 1st SS cleaved Insertion in IM by TIM 23 Also for proteins produced by mitochondrion 20 Figure 12-28 Molecular Biology of the Cell (© Garland Science 2008) Things to Consider... 1. Think about the utilization of energy (i.e. ATP or GTP hydrolysis) in nuclear and mitochondrial translocation. 2. How do nuclear pore complexes and mitochondrial translocator complexes differ? 21