Question

You have discovered a new transmembrane protein that resides in the ER. The molecular weight of the protein is approximately 100 Kd and it contains a hydrophobic transmembrane domain in middle of the protein amino acid sequence. You want to determine whether the protein is a Type I or Type II transmembrane protein. For the experiment, you are given the following reagents: cDNA construct for the protein, a cell line, a centrifuge, SDS-page apparatus, reagents needed for Western blot analysis and an antibody made against the N-terminus of the protein. Basic biochemicals and enzymes routinely used in cell biology research labs are also available to you. Briefly outline your experimental strategy for determining the topology of the protein and show expected Western blot analysis results if the protein is 1) Type I transmembrane protein or 2) Type II transmembrane protein.

Answer 1

A type I transmembrane protein is anchored on the memberane of the ER due to the presence of a sto transfer sequence in its N- terminal and type II transmembrane proteins are targetted and anchored by another signal sequence which is present on the C terminal of the protein . Hence the orientations of the to transmemberane proteins are different. The cDNA construct can be cloned into the cell line where the protein of interest will be expressed, The cells can then be lysed using detergents etc. and then centrifuged to separate the cell pellet from the supernatant containing the proteins, the proteins can then be extracted by salting out, dialysed and then run a native PAGE in the SDS PAGE apparatus so that the protein is not denatured due to reduction. We already know that the molecular weight of the transmembrane protein is 100kD. In SDS page the proteins in the cell lysate are seperated according to molecular weight and we can concentrate on the band corresponding to 100kD by comparing it with a standard protein marker hich is also run in the gel. The bands can then be transferred to a nitrocellulose membrane and western blotting can be done using the antibody specific for the N terminal anchor sequence that is provided. If the protein were to be a Type I transmembrane protein it will have a N terminal sequence that will bind to the antibody. Upon addition of a secondary antiibody a reporter attached to will give a band on the memberane. If it were to be a Type II transmembrane protein, it ill not have the N terminal equence but will have a C terminal sequence to which the provided antibody will not bind hence , the unbound antibody will be washed away and further no band will be observed upon addition of secondary antibody. The presence of band will confirm that the protein is type I and the absence will indicate that it is Type II.