26 Jun ErbB-2 is a receptor found on the plasma membrane of mammalian
Question
ErbB-2 is a receptor found on the plasma membrane of mammalian cells. Recent studies have shown that this protein can move from the plasma membrane to the nucleus. Because most proteins that shuttle between the cytoplasm and the nucleus are soluble and not integral membrane proteins, the mechanism by which ErbB2 undergoes this transport is of particular interest (see Giri et al., 2005, Mol. Cell Biol. 25:11005–11018.) In the first experiment, RNA interference (siRNA) is used to reduce levels of importin 1. Cells are either not subjected to siRNA (), or have their expression of importin reduced (imp), or are subjected to a nonsense siRNA (NS). The presence of ErbB-2 and two other proteins, tubulin and histone H3, in both cytoplasmic and nuclear fractions is then analyzed by immunoblotting using antibodies against each of these proteins. The data are shown on the immunoblot below in Figure (a). In the second experiment, lysates of cells expressing either wild-type (WT) ErbB-2 or mutant ErbB-2 lacking its nuclear localization signal (NLS) are immunoprecipitated with antibody to ErbB-2 or a control antibody (mIgG). The immunoprecipitates are then examined by immunoblotting with antibodies to importin 1 or to ErbB-2 as shown in Figure (b). a. Explain the role of importin proteins in nuclear transfer. (5 points) b. What do these data (part a) suggest about the relationship between ErbB-2 and importin 1 and why? (5 points) In further studies, cells were either untreated () or were induced to overexpress wild-type Ran (WT) or mutant Ran in which a glutamine at residue 69 is replaced with a leucine (Q69L). Ran Q69L hydrolyzes GTP poorly. The nuclear and cytoplasmic fractions are assessed by immunoblotting for the presence of ErbB-2, importin 1, tubulin, and histone H3, as shown at the left in Figure (c). The amount of Ran and ErbB-2 in the total cell lysates is also shown in Figure (c). c. Provide an analysis of the data for ErbB-2 seen in part c. (5 points) d. From the data provided, what model can you propose to explain the mechanism by which ErbB-2 is translocated into the nucleus? (5 points) Cells are treated with the drug leptomycin B (LMB) which inhibits the nuclear export protein Crm1, which is an exportin, and then analyzed for the localization of ErbB2, as shown by immunofluorescence in Figure (d). Regions of overlap between nuclear and ErbB-2 staining appear white, some of which are indicated by white arrowheads. In addition, nuclei are isolated at 0, 2, and 12 hours after LMB addition and assessed for the presence of ErbB-2, as shown in Figure (e). (d) e. What effect does LMB have on ErbB-2 localization and why? (5 points) f. What would you predict would happen if LMB were used in cells overexpressing Ran Q69L? (5 points)
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