weber uiuc edu 29 October 2007 MCB150 Lecture 27 Lecture 27 29 October 2007 Announcements More broken hand notes Back to the ER Still asking How do proteins get targeted to the final destinations o We know how it gets there o Without any other signal it will remain a soluble protein o What does this mean It is not attached to a membrane o All we have covered so far are soluble proteins What about transmembrane proteins o What about those targeted to the ER o Their destination is the plasma membrane or the membrane of an organelle like a lysosome the ER itself the golgi apparatus etc o Clearly need a different kind of signal Types of transmembrane proteins o Single pass transmembrane proteins Spans once N terminus in cytoplasm C terminus in lumen of ER Spans once C terminus in cytoplasm N terminus in lumen of ER o Multi pass transmembrane proteins N lumen C cytoplasm but multiple passes through the membrane DIAGRAM Not every multi pass protein will have seven spans or its N terminus in lumen It all depends upon the signals that are presented In the absence of any additional signal do the default That is keep doing what you were doing o It is acceptable to present another signal if it is logical o There are signals that give a variety of different information o Some signal sequences will cause the peptide to span the membrane multiple times Insertion into Membranes N C What if N terminus is in lumen and C terminus in cytoplasm with one span o Need a second signal to leave it in the membrane What got you to the ER The signal peptide Up to now this signal was at the amino terminus The fate of it is to be cut off by signal peptidase o This gets you to the ER If you don t tell the cell otherwise you keep translating the protein into the ER o This eventually will be a soluble protein in the lumen of the ER To stitch this into the membrane need to present a new additional signal o Need to insert into the membrane and close the translocon o A STOP transfer sequence 20 aminos or so in length and hydrophobic in nature o Hydrophobic amino acids makes sense because they will end up in the membrane This signal says Page 1 of 7 weber uiuc edu 29 October 2007 1 Eject from the translocon and make me a member of the membrane a transmembrane region of the protein 2 Close and dispose of the translocon DIAGRAM o Present the signal sequence and arrive at the ER o Push through translocon o The stop transfer sequence is presented o The signal on the amino terminus will still be popped off by signal peptidase The red ones in the diagram The signal peptide is then digested o The N terminus is in the lumen o The protein is transferred out of the translocon o The translocon closes disperses o The result is N lumen C cytoplasm Does the ribosome care about the translocon whether it is open or closed No It will not move off the translocon either closed or open until a STOP codon is encountered and the ribosome dissociates o It will continue to run through elongation o Because the translocon is closed the rest of the peptide is squirted out of the ribosome into the cytoplasm o In the absence of any additional sequences the rest of the amino acids added to the peptide will be added to the cytoplasm Result is N lumen and C cytoplasm Insertion into Membranes C lumen MCB150 Lecture 27 N cytoplasm To get this arrangement need to do something different So far the amino terminal sequence causes the entire protein to appear in the lumen where it is then cut off o To swap which end is in the lumen we could flip the entire transmembrane protein This is too difficult too hard to cause the protein to flip orientation o The best option is to build it correctly to begin with o But still need to move through the translocon How do we do this The first signal to be presented can be internal to the peptide o There is no reason the first signal cannot be internal to the peptide Internal signals are different from N terminal signal peptides o To avoid confusion he will refer to N terminal as amino terminal If you are somewhere other than the beginning of the protein the behavior is still the same the signal causes SRP binding which gets you into the translocon Departure of the SRP opens the translocon and the peptide gets threaded in in the middle leaving the N terminal end in the cytoplasm But in this case you do not chop off the N terminus o There is no way to just chop off the signal portion and reconnect the two pieces o If you remove the signal peptide you will remove those initial amino acids at the N terminus Do not want to lose those starting amino acids o Cannot afford to hydrolyze this signal peptide It stays in the membrane Page 2 of 7 weber uiuc edu 29 October 2007 MCB150 Lecture 27 If it isn t removed then it stays It is hydrophobic and will stay in the membrane o Because the orientation is different when translation is finished the Cterminus is in the lumen All the rest of the amino acids are in the lumen When the translation is finished the translocon disperses and the internal signal becomes the membrane spanning region o The rest of the peptide will be threaded into the lumen o BiP will pull the rest through Bip is there waiting to finish o Also the translocon is made up of three protein components so the peptide can be pushed through ejected to the side What is the first signal you present to the cell o EXAM Know how to recognize which is which the ones we are talking about now will stay in the membrane and will not be chopped off Everything else about this signal is the same as the previous o The only difference with this signal besides not being on the N terminus is that the signal will remain in the membrane o Since the N terminus is in the cytoplasm the C terminus will dangle into the lumen as long as there is no further modification For the purposes of MCB150 this is as complicated as we will make it o There is a figure in the text we are supposed to ignore See the web site for info on which figure o EXAM If the first presented signal is an amino terminal signal peptide then the N terminus dangles in the lumen of the ER o EXAM If the first presented signal is an internal signal peptide then the N terminus dangles in the cytoplasm o There are exceptions to these rules but for the exam and for our homework this is how we are to answer our questions Spanning the Membrane Multiple Times Just …