Question 1

 

 You have identified an ion channel protein the partial sequence of which is shown below. Based on the sequence, can you predict where and how many transmembrane domains this protein might have in this region?

  1.   
       
    40         50         60         70         80         90          
   
    LFYSTYGALY LSLGFNPIIE SLMTAFYFSI ETMSTVGYGD IVPVSESARL FTISVIISGI
   
    100        110        120        130        140        150
   
    TVFATSMTSI FGPLIRGGFN KLVKGNNHTM HRKDHFIVCG HSILAINTIL QLNQRGQNVT
   
    160        170        180        190        200        210
   
    VISNLPEDDI KQLEQRLGDN ADVIPGDSND SSVLKKAGID RCRAILALSD NDADNAFVVL
   
    220        230        240        250        260        270
   
    SAKDMSSDVK TVLAVSDSKN LNKIKMVHPD IILSPQLFGS EILARVLNGE EINNDMLVSM
   
    280        290        300
   
    LLNSGHGIFS DNDELETKAD SKESAQK

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  2. Suppose you have a k+ concentration of 140mM outside a cell and 4mM inside the cell. What is the Nernst potential across the membrane due to potassium?

     

  3. Using a technique known as patch clamp recording you can measure current flow through a membrane. Basically, a patch clamp works as follows: A glass recording micropipette is pressed against a cell membrane forming a tight seal. Recordings of current entering the pipette can be measured with the pipette attached to the cell. Normally, ions will not flow through membranes, so no current can be conducted. However, if some membrane proteins, such as ion channels, are captured in the tip of the micropipette, current can flow and be measured. As few as one, or as many as several hundred ion channels can be captured in a single experiment.

     

     

     

     

     

     

     

     

      

     

    Suppose that you observe the following current in a patch clamp experiment. What can you surmise about the flow of ions through the membranes? How do you explain the three different currents? How does the measured rate of ion flow compare with the diffusion limit of ion flux (the diffusion limit is ~107-108/sec). (The charge of one electron is 1.06x10-19 coloumbs.)

 

 

 

 

 

 

 

 

 

 

 

  • You have a scorpion toxin that works by binding to and blocking potassium channels. Some potassium channels, however, are not affected by the scorpion toxin. You have the cDNA encoding for a potassium channel which is susceptible to the scorpion toxin. By injecting the mRNA into amphibian oocytes you can get expression of the protein and measure current using the patch clamp technique. (You can set up conditions such that the oocyte’s native ion channels are not functional.) How might you use the available reagents to determine the orientation of the ion channel in the membrane (determine the extracellular and intracellular domains)?

     

     

  • You would like to clone a sodium channel from a new strain of bacterium. Attempts to clone by homology to known ion channels are not successful. Using techniques described above, can you devise a strategy for cloning the sodium channel?

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    Question 2

     

     

    The signal recognition particle (SRP) consists of six polypeptide subunits. After protein synthesis is initiated in the cytoplasm, the 54 kDa subunit of the SRP binds to the signal sequence shortly after it is synthesized on the ribosome. This SRP-ribosome unit then associates with the SRP receptor (docking protein) on the endoplasmic reticulum (ER) where synthesis continues and the newly synthesized protein is translocated across the ER. (See pp681-694)

    In experiments designed to sort out the factors necessary to promote translocation, a complete cell-free translation system was incubated with a mRNA encoding a secretory protein of 45 kDa when fully modified, [35S]methionine to monitor protein synthesis, and various preparations containing different factors, as indicated below. GMP-PNP and AMP-PNP are non-hydrolyzable analogs of GTP and ATP respectively. After each of the preparations was incubated with the translational system in appropriate buffers, the sample was incubated with a protease (proteinase K). Then all proteins were precipitated, denatured and separated on a SDS gel. Autoradiography of the gel revealed the pattern shown.

     

     

     

     

     

     

     

     

     

     

     

     

    Lane

     

     

     

     

     

     

    1

     

    SRP-Ribosome complex

     

    Microsomes

     

    ATP

     

    GTP

     

     

    2

     

    SRP-Ribosome complex

     

    Microsomes

     

    AMP-PNP

     

    GTP

     

     

    3

     

    SRP-Ribosome complex

     

    Microsomes

     

    ATP

     

    GMP-PNP

     

     

    4

     

    SRP-Ribosome complex

     

    Microsomes

     

    ATP

     

    GTP

     

    Additional time

     

    5

     

    SRP-Ribosome complex

     

    Microsomes

     

     

     

     

    6

     

    SRP-Ribosome complex

     

    Microsomes

     

    ATP

     

     

     

    1. What is the significance of the discrete bands in lanes 1, 3 and 4 of the autoradiogram and of the diffuse bands in the other lanes?

      2.  

    2. What can you conclude from this experiment about the factors required for protein translocation and the mechanisms involved in this process?

     

     

     

    Question 3

     

    1. You have identified a novel ER membrane protein in yeast. You are interested in determining what region of the protein is important in targeting the protein to the ER membrane. How might you accomplish this?

       

    2. The sequence you identify in part A turns out to consist of two lysine residues at positions &emdash;3 and &emdash;4 from the C-terminus. A similar sequence has been identified in mammalian type I transmembrane proteins. Intrigued by the similarity, you decide to take a genetic approach to investigating the machinery involved in retrieval of type I transmembrane proteins to the ER. Using techniques covered in the early sections of this class, you have isolated a series of temperature sensitive (ts) mutants defective in retrieval. The ts mutants fall into two classes: ret1 for retrieval defective 1 and sec21. The sec21-1 allele of this gene was originally identified on the basis of a defect in secretion.

      To quantify the effects of these mutations, you make a fusion protein containing the dilysine (diK) retrieval sequence. If this diK fusion protein is retrieved, it is not exposed to a post-ER protease. The fusion protein is expressed in yeast cells incubated with radiolabelled amino acid, run on SDS-PAGE and detected by autoradiography. The results for three mutants are shown below. In the absence of retrieval, the diK-fusion protein is processed by the post-ER protease. Based on this data, do all three mutations have a similar effect on processing of the diK-fusion protein?

       

       

       

       

       

       

       
    3. To quantify the effects of these mutations on secretion, you analyze processing of carboxypeptidase Y (CPY) by a similar approach. CPY exists in two precursor forms termed p1CPY and p2CPY, which are present n the ER and Golgi complex respectively. The mature CPY (mCPY) is present in the vacuole. The autoradiograms are shown below. Based on this data, do all three mutations have a similar effect on secretion of CPY to the vacuole?/OI>