Chapter 13 Moving Proteins into Membranes Organelles 2 Processes for Protein Targeting 1 Signal Based Targeting every protein has a signal short specific AA sequence that directs protein where to go and it directs the protein to a particular organelle Used for proteins designed to go the Golgi mitochondria ER nucleus peroxisomes chloroplasts 2 Vesicle Based Targeting designed for proteins that get secreted from the cell Go to the plasma membrane or lysosome Targeting of Proteins To Across the ER Membrane Pathway for making sorting ER Golgi lysosomal proteins are the same Rough ER ribosomes don t stay permanently attached ER signal sequence located within 16 30 AA at the N terminus directs protein to the ER Core Sequence within the ER SS 6 12 hydrophobic AA usually preceded by 1 or 2 positively charged AA s Directs the protein to go into the ER If you put this sequence in any protein it will go to the ER To target the ER membrane need a signal recognition particle SRP and its receptor P54 signal recognition protein What will bind to the ER signal sequence at the core sequence On the ER membrane there are SRP receptors Translocon translocation channel allows proteins to cross Slide 4 graphic Insertion of Proteins into the ER Membrane Single Pass Proteins go through the membrane once Type I II III Type I Protein N terminus in the ER lumen o Uses SRP and SRP receptors o N terminus where ER signal sequence located o Second signal needed to make the protein stick in the ER membrane instead of going all the way through to the lumen Stop Transfer Anchor Sequence 20 25 hydrophobic AA o will become the transmembrane domain o o C terminus remains outside the cytoplasm forms an alpha helix that is inserted into the membrane Type II III have internal hydrophobic signal anchor sequence that becomes the transmembrane domain also 20 25 hydrophobic AA that forms helix o Brings the RNA protein ribosome complex directly to the Sec61 translocon Slide 8 highlights the differences between the types Multi Pass Proteins go through the membrane multiple times Type IV Must have as many signals as there are transmembrane domains o Alternate between stop transfer anchor STA and signal anchor SA Type IVA N terminus in cytoplasm glucose transporters ion channel proteins Types IVB C terminus in cytoplasm G protein coupled receptors Hydropathy Profiles based on a hydropathic index given to each AA Hydrophobic positive number Polar Charged negative number Transmembrane domains are 20 25 AA that are hydrophobic o Use graph to see if you have transmembrane domains or not Protein Modification and Folding in the ER 1 Glycosylation addition of sugar carbohydrate groups O linked chains carbs attached to OH in a serine or threonine o Tend to be very small 1 4 sugars and rare occurrence N linked Oligosacharides attached to asparginine o More common larger and can have branching structures 2 Disulfide Bonds form 3 Multisubunit proteins come together 4 Proteolytic Cleavage of some proteins Precursor transferred from dolichol carrier to an Asn X Ser or Asn X Thre 12 sugar precursor that initially adds to proteins in the ER Before if leaves the ER have removal of 3 glucose molecules usually 1 mannose o It confers some stability at first but then must be removed Sorting of Proteins to Mitochondria Each organelle has different sequence that directs proteins to specific subcompartments Mitochondrial Matrix Targeting Sequences amphipathic located at the N terminus 30 50 AA Forms an helix One side has lots of charged AA s lysine arginine and other side of helix has hydrophobic Proteins are fully translated in the cytoplasm and remains unfolded Tom20 22 binds the matrix targeting sequence on the outer membrane Tom40 translocon on outer membrane Tim44 translocon on the inner membrane ATP is used to pull the protein through the inner membrane to the mitochondrial matrix Targeting Proteins Location Matrix Inner Membrane A Inner Membrane B Intermembrane Space Imported Protein Alcohol Dehydrogenase III Cytochrome oxidase subunit ATP synthase subunit 9 Cytochrome b2 Location of Targeting Sequences MTS at front Single Pass Protein Multipass Proteins 2nd cleavage in intermembrane space For Inner Membrane Path A uses same machinery for targeting matrix proteins 1 Binds to the Tom20 22 receptor 2 Opens the Tom40 translocon to pass through outer membrane 3 Opens Tim23 translocon to pass through inner membrane 4 Stop transfer prevents further movement through translocon o Protein slides laterally out of the membrane o Protein allowed to stay in the inner mitochondrial membrane Path B have matrix targeting sequence internal hydrophobic domains recognized by Oxa1 Has multiple internal sequence and will all will become transmembrane domain 1 Binds to Tom20 22 2 Opens translocon Tom40 3 Goes through Tim23 17 4 Oxa1 additional translocon that recognizes oxa1 targeting sequences become transmembrane domains For Intermembrane Space Path A predominant proteins carry 2 different N terminal targeting sequences 1 Matrix Targeting Sequences bind to Tom20 22 receptor 2 Opens Tom40 translocon 3 Goes through Tim23 17 translocon But because AA sequence is different it is recognized by protease and degraded Initially acts like transmembrane domain by sliding laterally o Sequence released into intermembrane space Targeting of Peroxisomal Proteins Peroxisomal matrix proteins have C terminal peroxisomal targeting sequence with Ser Lys Leu at the end Protein completely translated and folded in tertiary structure Pex5 receptor recognizes peroxisomal targeting sequence and bind Pex5 receptor goes into the peroxisomal matrix with cargo protein Peroxisome has different pH of 5 vs cytosol pH of 7 o When protein goes into peroxisomal matric pH difference causes there to be a change in affinity of Pex5 receptor and releases the cargo protein Transport Into and Out of the Nucleus Nuclear Pore Complex NPC is made of nucleoporins hydrophobic making lining of channel hydrophobic Nucleus doesn t have translocons so NCP provide channels for things to go into out of the nucleus Nuclear Localization Sequences NLSs has 7 basic AA at the C terminus Pro Lys Lys Lys Arg Lys Val Proteins are already translated and folded Importin binds to the NLS Importin on outside lots of hydrophobic amino acids o Interacts with FG nucleoporins that line nuclear pore Once inside the nucleus importin will interact with Ran Ran with GTP has nigh affinity for importin and binds to it changes