weber uiuc edu 26 October 2007 MCB150 Lecture 26 Lecture 26 26 October 2007 Announcements More broken hand notes Turn off your cell phones or die Where We Left Off Proteins being imported into a mitochondria o Can be inserted into the outer or inner membrane o Without another targeting signal you will end up in the matrix E g Kreb s cycle enzyme mitochondrial RNA polymerase or mitochondrial ribosomal protein Lots of examples to get into the matrix o Tom translocator of outer membrane Presquence is recognized by some of the Tom proteins Need ATP to ratchet into Tom o Tim translocator of inner membrane Drawn across Tom and into Tim Cross as much of intermembrane space as needed Emerge into matrix side of Tim Once you emerge into the matrix there are mitochondrial Hsp70 s that will grab you and ratchet you in to the mitochondrial matrix o This too uses ATP as an energy source Now we are in the mitochondria The mitochondrial processing peptidase MPP will remove the mitochondrial pre sequence o The pre sequence has done its job and is not needed any more o It gets cut off of the amino terminus o MPP will leave behind two pieces the rest of the peptide protein and a piece of pre sequence that will be chopped up for its amino acids The protein will either be folded right away or will need assistance in folding For those that can be folded a separate set of chaperone proteins help The purple beads on the blue string of protein DIAGRAM are helping the protein fold right away A good number of the proteins will need a lot of help to fold properly o The free chaperons will actually move the protein to a gigantic folding machine complex called a chaperonin The pre sequence has already been removed o Chaperones are individual polypeptides that function as unique chaperone molecules o Chaperonin complexes are large multimeric subunit complexes Chaperonins in mitochondria have 14 polypeptide chains o They exist to provide a location for the incoming protein to come in and fold the right way the first time o Only the protein and not the chaperones accompanying it will be permitted inside which takes some energy Need to remove the chaperons which takes energy The energy takes ATP ADP hydrolysis Page 1 of 7 weber uiuc edu 26 October 2007 MCB150 Lecture 26 Once inside the top door closes creating an isolated environment the protein folds then a trap door opens to let the protein out o The opening of the door requires energy ATP ADP as well MPP exists in the matrix of the mitochondria o Some proteins do not make it into the matrix These retain their presequence o Those proteins that are inserted into the outer membrane do not have their pre sequences removed because they are not exposed to any proteases like MPP to have the pre sequences cut off o You do not have to remove the pre sequence but if you make it to the inner matrix you will lose it the pre sequence In the diagram there are only four ATP requirements o Yet there are at least 5 energy requiring steps just to get the peptide chain into the mitochondria and in its final folded step Into Tom ATP Through Tom into Tim Out of Tim ATP Transfer to into chaperonin complex ATP Out of chaperonin complex ATP o Why is there no ATP requirement to move the protein into the intermembrane space Remember there is no ATP generation done by the ETC The mitochondrial matrix is more negative and the matrix space is more positive The negative charges in the matrix therefore draw the positively charged pre sequence into Tim No more energy is needed to draw the protein into Tim because the protein pre sequence is naturally positive and attracted to the negative region o The protein taps into the electrochemical gradient set up by the electron transport chain No ATP is spent for this As long as protons are pumped there will always be an electrochemical gradient o Just as the ETC powers oxidative phosphorylation the ETC powers the movement of the peptide chain into Tim via charge attractions Signals The default behavior is to stay where you are without a targeting signal o In the absence of a signal proteins in the cytoplasm stay in the cytoplasm o To get into mitochondria display a mitochondrial pre sequence o If you need to get into the nucleus you need an NLS What about other final destinations For example outside of the cytoplasm into a lysosome into the extracellular matrix etc Yes there are signals for these transports as well The bigger picture is how do proteins get where they need to Site of Translation The site of translation determines the fate of the protein Ribosomes are either free floating or bound to the ER in the rough ER Intact ribosomes only exist where translation is occurring Page 2 of 7 weber uiuc edu 26 October 2007 MCB150 Lecture 26 o You don t bring the subunits together until you have identified an initiator codon brought in the first charged tRNA laid in initiator factors and brought in the large ribosomal subunit Review from Unit 2 o You never form an intact ribosome unless you are in the process of translation ready to do protein synthesis If the ribosome is membrane bound then it would make sense that translation is occurring at the membrane o If there are ribosomes there then we can assume translation is occurring there What are the fates of the different proteins with respect to the ribosome o Free ribosomes in the cytosol make proteins that can end up in the nucleus peroxisomes chloroplasts and mitochondria These are where you end up if you display a targeting signal Without a targeting signal the protein ends up in the cytosol o Membrane bound ribosomes make proteins that can end up in the lysosomes secretory vesicles golgi apparatus the plasma membrane or those exported into the extracellular space When displaying a signal that takes you to a membrane the proteins will stay there Otherwise you stay in the membrane itself Experiments show that any protein exported outside the cell will be translated on a membrane bound ribosome There are steps along this pathway and members of those steps will also be synthesized by membrane bound proteins E g Secretory vessels will be filled with material translated by membrane bound organisms Same with proteins bound as integral proteins the golgi ER lysosomes extracellular space etc If those locations are your fate you ll be translated by a membrane bound ribosome o These are your two choices The Pulse Chase Experiment How do we watch a set of proteins making its way through the cell How can we