Published on October 12, 2007
Organelles and Protein Sorting: A protein’s fate in the cytosol A protein’s fate in the ER Transport carriers: membrane fission and fusion Protein sorting in the exocytic pathway Protein Sorting in the endocytic pathway Organelles and Protein Sorting Christine Suetterlin firstname.lastname@example.org, 824-7140 Slide2: General requirements for protein sorting: the signal hypothesis Protein import into the nucleus, mitochondria and peroxisomes Protein import into the ER Lecture I A protein’s fate in the cytosol Slide3: What determines the identity of an organelle? How is organelle-specific protein and lipid composition achieved? Slide4: Protein sorting in the cytosol establishes organelle identity What is required for protein sorting? : What is required for protein sorting? a signal (address), intrinsic to the protein a receptor that recognizes the signal and which directs it to the correct membrane a translocation machinery energy transfer the protein to its new place Slide6: necessary: It should be able to direct a protein into a new, membrane enclosed environment (lead to the protein’s protection from proteases) sufficient: it should be able to direct a cytosolic protein to the new environment It should specifically interact with a binding partner/receptor It should be removed from the protein upon arrival in target location What is expected of a sorting signal? Slide7: Press Release: The 1999 Nobel Prize in Physiology or Medicine NOBELFÖRSAMLINGEN KAROLINSKA INSTITUTET THE NOBEL ASSEMBLY AT THE KAROLINSKA INSTITUTE 11 October 1999 The Nobel Assembly at Karolinska Institutet has today decided to award the Nobel Prize in Physiology or Medicine for 1999 to Günter Blobel for the discovery that "proteins have intrinsic signals that govern their transport and localization in the cell" Summary A large number of proteins carrying out essential functions are constantly being made within our cells. These proteins have to be transported either out of the cell, or to the different compartments - the organelles - within the cell. How are newly made proteins transported across the membrane surrounding the organelles, and how are they directed to their correct location? These questions have been answered through the work of this year’s Nobel Laureate in Physiology or Medicine, Dr Günter Blobel, a cell and molecular biologist at the Rockefeller University in New York. Already at the beginning of the 1970s he discovered that newly synthesized proteins have an intrinsic signal that is essential for governing them to and across the membrane of the endoplasmic reticulum, one of the cell’s organelles. During the next twenty years Blobel characterized in detail the molecular mechanisms underlying these processes. He also showed that similar "address tags", or "zip codes", direct proteins to other intracellular organelles. Slide8: Ribosome assembly requires bidirectional nuclear transport Slide9: Bridges the inner and outer membrane 8-fold rotational symmetry 3 concentric rings complex (a least 30 proteins) 3000 to 5000 nuclear pores/cell site of protein and RNA transport into and out of the nucleus Slide10: How is nuclear transport regulated? Asymmetric import/export cycles, Cargo-receptor interaction depend on their environment Slide11: The regulation of a small GTPase GTP binding protein Small monomeric (not heterotrimeric G-Proteins) 2 different states (GTP-bound and GDP bound) different nucleotide binding state is regulated by GAPs and GEFs localization of GAPs and GEFs is critical for many reactions Guanine exchange factor GTPase activating protein Slide12: What happens if the nuclear localization signal is mutated? A mutation in the nuclear localization signal leads to cytosolic localization of the protein. The protein T-antigen with a wild-type NLS localizes to the nucleus Protein import into mitochondria: Protein import into mitochondria Proteins are imported into 4 distinct mitochondrial compartments Mitochondrial matrix Intermembrane space Inner membrane Outer membrane Slide14: How can mitochondrial protein import be studied? Protein import into mitochondria leads to 1. protease protection and 2. change in size due to removal of the import sequence In vitro reconstitution Chimeric proteins reveal the mechanism of protein import into mitochondria: Chimeric proteins reveal the mechanism of protein import into mitochondria Chimeric proteins Variable spacer region allows protein to be inserted in a defined manner: 1. crosslinking was used to identify component of the channel 2. proteins have to be unfolded for import into mitochondria 3. energy is required (ATP and a proton gradient) 4. import occurs where inner and outer membrane are close Slide16: How proteins are imported into mitochondria Sorting signal (for mito matrix): N-terminal amphiphatic helix Cytosolic chaperones keep the protein in an unfolded state and bring it to the translocation machinery Translocation machinery: TOM complex in Outer membrane (all proteins), TIM complexes in Inner membrane (specific subsets of proteins) signal sequence is removed by signal peptidase upon arrival in matrix and the protein folds Mitochondrial chaperones help with folding How are proteins imported into the intermembrane space?: How are proteins imported into the intermembrane space? Slide18: Protein import into peroxisomes The Pex/Peroxin import machinery is only responsible for peroxisomal matrix proteins, not for membrane proteins Slide19: How would a mutation in the peroxisomal import machinery affect peroxisome biogenesis? A mutation in a component of the peroxisomal import machinery (peroxins) leads to cytosolic distribution of peroxisomal matrix proteins, but not of peroxisomal membrane proteins Wildtype situation Mutation in Peroxin import machinery Peroxisomal membrane protein Peroxisomal matrix protein (Catalase) Slide20: Protein trafficking pathways The same pool of ribosomes synthesizes cytosolic and secretory proteins. Organelle sorting signals: Organelle sorting signals Nucleus Internal import: One cluster of 5 basic amino acids, or two smaller clusters of basic residues separated by ≈10 amino acids export: Leucine rich: eg LQLPPLERLTL (rev protein of HIV-1) Mitochondrion N-terminal 3 – 5 nonconsecutive Arg or Lys residues (--> amphiphatic helix) often with Ser and Thr; no Glu or Asp residues Chloroplast N-terminal No common sequence motifs; generally rich in Ser, and Thr and small hydrophobic amino acids, poor in Glu and Asp residues Peroxisome C-terminal Usually Ser-Lys-Leu at extreme C-terminus ER N-terminus hydrophilic domain (often basic) followed by 6 to 12 hydrophobic residues Internal 16 to 30 hydrophobic residues Slide22: Dobberstein and Blobel, 1975 Experiments with microsomes led to proposing the signal hypothesis ER-bound ribosomes synthesize proteins that are translocated into the ER and thus protease protected. Mechanism of ER translocation: Mechanism of ER translocation Slide24: What directs a protein into the ER? SRP and its receptor: SRP and its receptor SRP 6 protein subunits of which one has GTPase activity, 1 RNA subunit (300 bases) SRP-receptor 2 subunits 1 is transmembrane (), 1 is peripheral and has GTPase activity () Slide26: Multimeric protein complex that creates an aqueous channel in the ER membrane Permeability has to be tightly regulated--> plug conserved mechanism The translocon How can a component of the translocation pathway be isolated?: How can a component of the translocation pathway be isolated? Translocon TRAM: 6 TM domains Sec61TM domains Sec61: 1 TM Sec61: 2 TM aqueous channel Gorlich et al., Nature, 1992 The same strategy was applied for the isolation of components of the mitochondrial translocation machinery (Tokatlitis et al. Nature, 1996) The translocon is conserved: The translocon is conserved Mammalian cells yeast bacteria Sec61 Sec61p SecY Sec61 Sbh1p SecG Sec61 SSs1p SecE Energy requirements for ER translocation: Energy requirements for ER translocation Unfolding the protein in the original location co-translation translocation: chain elongation during translation post-translational translocation/mitochondrial import: chaperone (Hsp70) unfolds protein in an ATP-dependent manner Opening of the “gate” mutual stimulation of GTPase activities of an SRP subunit (p54) and the subunit of SRP-receptor Pulling through the channel: chaperone activity inside the target organelle (Hsp70) that in addition helps fold the protein Molecular chaperones: Molecular chaperones Up-regulated during heatshock, conserved 2 classes Hsp70: protect a misfolded or unfolded protein from degradation/folding, Hsp40 and Hsp90 as cofactors Hsp60 (chaperonin), actively helps protein folding Organelle specific, e.g. Bip in the ER
Information about 231B 2006 Suetterlin Lec1. Entertainment. protein organelles. Published on October 12, 2007. Author: Haggrid. Source: authorstream.com.