weber uiuc edu 24 October 2007 MCB150 Lecture 25 Lecture 25 24 October 2007 Announcements More broken hand notes Weekly Review Question 1 o Read the question carefully o Careful about 90 of us have it wrong o Don t forget what we learned last week and read all of the options One of the statements needs to be thought about carefully o ANSWER The statement that was tripping people up was this one T F If the gene prim codes for primase and a cell is prim the cell is wild type with regards to transcription The answer is true because primase isn t used for transcription Pffft o He doesn t ask truly cumulative questions but our prior knowledge of what is going on should have helped us answer this Material Transport Cytoplasm Nucleus Recap we are talking about how material gets from one side of the envelope to the other On the table now is how does it go from the cytoplasm to the nucleus Because the default behavior is to stay in the cytoplasm where the protein is made the cell has to go out of its way to move the molecule in for larger molecules o Need to tell the cell where you need to go Therefore the protein needs a signal to indicate the need for transport into the nucleus We ll talk about lots of these over the next few lectures o Ribosomal proteins to be incorporated into a subunit o Transcription factors o DNA polymerase o Etc Lots of reasons proteins need to get into the nucleus NLS nuclear localization signal o A sequence of amino acids either continuous or discontinuous regions as long as it folds properly o With either it has to be interpreted and needs to be recognized appropriately The carrier protein molecule is called an importin o It imports the protein molecule o Our frame of reference here is from within the nucleus Thus something being imported is being brought from the cytoplasm to the nucleus and something being exported is leaving the nucleus and going to the cytoplasm o The importin will localize the protein to the nucleus The importin binds to the NLS via a protein protein interaction o This is a productive interaction The cytoplasmic filaments recognize the importin attached to the cargo molecule and allow both in DIAGRAM The figure is stripped down to cover what we need to know for this class Page 1 of 9 weber uiuc edu 24 October 2007 MCB150 Lecture 25 o Note the figure is not drawn to scale It is grossly exaggerated w r t the protein importin complex o The importin cargo complex is not huge with respect to the pore complex The approach of the importin and cargo starts a cascade of events at the filaments to allow the protein to be pulled in with the importin o They are pulled and pushed through the pore complex to the other side There is an event that causes the importin to release the protein cargo o We do not need to know exactly how this happens for MCB150 Once it lets go the cargo protein is now where it needs to be DIAGRAM The purple stretch on the protein is the NLS o KNOW THE NLS IS PART OF THE PROTEIN S SEQUENCE o It is NOT a separate protein Once in the nucleus and the importin has released its protein cargo it needs to get back to the cytoplasm to grab another protein o Traffic across pore complexes is bi directional There are not some nuclear pore complexes that bring things in others out Whoever gets there first gets to cross The importin molecule is taken back out to the cytoplasm Taking the importin across is an energy dependent process o Does it take energy to bind an importin to a NLS No o Does it take energy to approach or cross the pore complex No Do not be tricked It does not require energy surprisingly It is not transport that costs energy o Does it cost energy to cause the importin to let go No o Does it cost energy to move the importin back to the cytoplasm No o Does it take energy to restore the conformation of the importin YES It needs to be recycled back to a 3D conformation capable of accepting a new cargo molecule How was it forced into letting go of the cargo protein A conformation change Until it is put back it cannot bind to the next NLS o GTP is used as an energy source to recycle the importin molecule to restore the 3D conformation of the importin so it can bind to the next cargo protein We have now completed the coverage of how proteins get into the nucleus What Keeps Nuclear Proteins Out of the Cytoplasm What keeps proteins and RNA in the nucleus Why don t you find RNA polymerase out in the cytoplasm o Remember the default behavior is to stay where you are Why doesn t the signal ship it back out Because the signal is not correct for export The signal is for import only The signal presented is important if the correct appropriate signal isn t present then no action is taken There will have to be a molecule that recognizes the signal binds and takes the cargo out In 1995 two proteins were found to have a string of amino acids that allow the export of proteins Page 2 of 9 weber uiuc edu 24 October 2007 MCB150 Lecture 25 NES Nuclear Export Signal o It gets the protein from the nucleus out into the cytoplasm o It is a totally separate stretch of amino acids from the NLS Once again the amino acid sequence is necessary and sufficient to get you to the cytoplasm o Necessary the sequence is needed to transport a protein to the cytoplasm o Sufficient that simple sequence is enough to get any protein out of the nucleus and into the cytosol Add it to another protein that normally shouldn t be exported and that protein will be pushed out What is unusual about that sequence o Usually if you see something that is unusual in biology then it probably means something specific o In this case the same type of amino acid is showing up more times than statistically expected o About half of the amino acids are leucines o NES s are leucine rich o There is something that happens between the NES and the carrier o IT IS NOT IMPORTANT TO MEMORIZE THE NES or NLS SEQUENCE o EXAM INSTEAD KNOW THE OVERALL CHARACTERISTICS WHAT IS RICH IN WHAT AN NES IS RICH IN LEUCINE Exportins A carrier or shuttle protein that takes proteins out of the nucleus through a nuclear pore complex is called an Exportin The exportin recognizes an NES and forms a cargo complex to export the protein out into the cytoplasm DIAGRAM The diagram makes it look like a separate protein slapped on It is not o The NES is not slapped on to the protein as an extra thing it is part of the protein …