Cytoplasm chromosome Periplasm Inner membrane Outer membrane Outside the cell BACTERIAL CELL mRNA protein MIT Biology Department 7.012: Introductory Biology - Fall 2004 Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel EUKARYOTIC CELL Mitochondrion Plasma membrane Nucleus Endoplasmic Recticulum Golgi Apparatus Cytoplasm Outside the Cell N Eukaryotic Cell Bacteria Insulin Growth Factors Antibodies Insulin Receptor Growth Factor Receptors Histidine synthesis Lactase Glycolysis Enzymes Cyclins ToxinLactose Receptorg2g1galactosidase Fully Secreted Protein (Outside the Cell) Membrane Protein Cytoplasmic Protein Examples George Palade Images removed due to copyright reasons. Images removed due to copyright reasons. Hamster pancreatic Nucleus Mitochondrion Earliest Time point Next observed location Location After Golgi Millstein “in vitro synthesis of immunoglobulin light chains. … To our delight we ran into the unexpected observation of the existence of a biosynthetic precursor of light chains. Further experiments led us to propose the extra N-terminal sequence was a signal for vectorial transport across the membrane during protein synthesis. That was the first evidence which indicated that the signal for secretion was an N-terminal segment, rapidly cleaved during protein synthesis.” FROM NOBEL LECTURE 1984 Image removed due to copyright reasons. Cytoplasmic Extracts N Messenger RNA Ribosomes & charged tRNAs Microsomes (RER vesicles) in vitro Blobel Image removed due to copyright reasons. + added late + + + added early -- Purified extract ++- Microsomes +++ Message Ribosomes tRNAs Protein in supernatent Protein in lumen of microsomes Protein in supernatent N Protein in supernatent From the previous experiment, Blobel demonstrated that the amino acid sequence at the beginning (N terminus) of exported proteins is recognized by a complex. This complex is required to get the protein into the lumen of ER. To get into the lumen of the ER the protein has to be just beginning to be translated. Since not all exported proteins have the same N terminus, Blobel predicted, like Millstein, whatever the sequence was, it would be later cleaved. Gunter Blobel Nobel Laureate, 1999 Image removed due to copyright reasons. + Hydrophobic amino acids N ~20 amino acids 5’ “Signal Sequence” Bacterium Eukaryotic Cell N N N SRP Signal Sequence Signal Recognition Particle SRP receptor “Docking Protein” SRP receptor “Docking Protein” Translocon SRP receptor “Docking Protein” SRP receptor “Docking Protein” Signal peptidase cleaves off the signal SRP receptor “Docking Protein” Fully secreted Protein SRP receptor “Docking Protein” Signal peptidase cleaves off the signal SRP receptor “Docking Protein” Membrane protein EUKARYOTIC CELL Plasma membrane Endoplasmic Recticulum Golgi Apparatus Cytoplasm Outside the Cell Transport Vesicles EUKARYOTIC CELL Plasma membrane Endoplasmic Recticulum Golgi Apparatus Cytoplasm Outside the Cell EUKARYOTIC CELL Plasma membrane Endoplasmic Recticulum Golgi Apparatus Cytoplasm Outside the Cell EUKARYOTIC CELL Plasma membrane Endoplasmic Recticulum Golgi Apparatus Cytoplasm Outside the Cell Secretory Vesicles Plasma membrane Endoplasmic Recticulum Golgi Apparatus Cytoplasm Outside the Cell Secretory Vesicles Plasma membrane Endoplasmic Recticulum Golgi Apparatus Cytoplasm Outside the Cell Plasma membrane Endoplasmic Recticulum Golgi Apparatus Cytoplasm Outside the Cell Plasma membrane Endoplasmic Recticulum Golgi Apparatus Cytoplasm Outside the Cell Plasma membrane Endoplasmic Recticulum Golgi Apparatus Cytoplasm Outside the Cell Plasma membrane Endoplasmic Recticulum Golgi Apparatus Cytoplasm Outside the Cell Transport vesicles Secretory Vesicles cytoplasm chromosome Periplasm Inner membrane Outer membrane g2-galactosidase lacZ How were the Sec genes identified? Bacterium mRNA Cytoplasm chromosome Inner membrane g2-galactosidase lacZ How were the Sec genes identified? Active g2-galactosidase is a tetramer. This cell can utilize lactose as a carbon source. LAC+ Gene encoding Exported Protein lac Z gene 5’ 3’ 5’ 3’ 5’ 3’ Gene Fusion The 5’ end of the coding region of lac Z is fused to the 5’ end of a gene encoding an exported protein including the signal sequence. ‘lac Z gene 5’ 3’ Gene Fusion Where the 5’ end of the lac Z gene is fused to the 5’ end of a gene encoding an exported protein including the signal sequence. This gene fusion results in a hybrid protein where the N-terminus of g2-Galactosidase is fused with a signal sequence. Signal Sequence N C g2-galactosidase Cytoplasm chromosome Periplasm Inner membrane Outer membrane Hybrid protein Gene fusion The hybrid protein protein localizes to the membrane. This cell is unable to utilize Lactose as a Carbon Source. LAC - Cells with this gene fusion are… Cytoplasm chromosome Periplasm Inner membrane Outer membrane Gene fusion LAC- LAC+ >95% of the Lac+ mutants have mutations ….. linked to the gene fusion resulting in ….? X Hybrid protein X X X X Jon Beckwith + Hydrophobic amino acids N ~20 amino acids C X Cytoplasm chromosome Periplasm Inner membrane Outer membrane Gene fusion Hybrid protein LAC- LAC+ X X X X Cytoplasm chromosome Periplasm Inner membrane Outer membrane Hybrid protein Gene fusion LAC- LAC+ X X X X Conditional Lethal How to get get Mutations in essential genes Temperature- sensitive Cold-sensitive 20°C 37°C Active Inactive Active Conditional Lethal How to get get Mutations in essential genes Temperature- sensitive Cold-sensitive 20°C 37°C Active Inactive Inactive Active Sec A Sec B Sec D Sec E Sec G Sec Y Post- Translational Post- translational Cotranslational Translational state of protein in channel What is the Energy Source? How does the protein cross the membrane? Signal Destination GTP hydrolysis ATP hydrolysis Powered by translation Importins deliver to Nuclear Pore Complex (NPC) Chaperones bind Protein enters Mito. Channel SRP binds SS SRP binds DP Protein enters channel Nuclear Localization Signal (NLS) 7aa + charged N-terminal Amphipathic Helix 20-50 aa Cotranslational Powered by translation SRP binds SS SRP binds DP Protein enters channel Signal Sequence Signal Sequence NucleusMitochondrion Outside the cell Plasma membrane NOYesYesYes Signal Cleaved?