Question

5. (9pts) You are studying an ER membrane protein, YFG1 (outlined in yellow), with three transmembrane domains (rectangles A, B & C at the amino acid positions according to the following map, connected by soluble domains (wavy lines): N-term 10 10 Com Amino acid position (not to scale) 30 60 80 140 160 310 ABCI YFG1 alkaline phosphatase To determine how the protein is arranged in the ER membrane, you construct a series of shortened YFG1 proteins that are connected at the C-terminus to another reporter protein, alkaline phosphatase (AP, blue bar). Your protein constructs 1-6 are shown below, where the yellow bar shows the amino acids left from the YFG1 protein, and the blue bar shows AP. Assume that AP activity will be high if it is located in the cytoplasm, and that AP activity is low if it is located in the ER lumen. alkaline phosphatase activity original contracts W N-term W C -term HIGH - HKSH A. Draw a predicted structure of Construct 6 (full-length YFG1) in the following diagram of the ER membrane. Your drawing should contain boxes with letters and wavy lines to represent YFG1 and a label for the location of AP Cytoplasm Construct 6 ER membrane ER lumen B. In Part A, add the flanking charges around transmembrane domain A that would lead to your predicted protein orientation. C. Please explain how the protein structures of Constructs 1-6 in the ER membrane lead to the observed activities of alkaline phosphatase. 30397 IN SA D. In a follow-up experiment, you take Construct 6 and remove amino acids 55-85. You observe that the activity of alkaline phosphatase changes from HIGH to LOW. Please explain this observation referring to the structure of the protein. explain this ou

Answer 1

(A. cytoplasm CRplam FR lumen

C. construct 1 - the activity of alkaline phosphatase (AP) is high as the construct's N-terminal lies in ER lumen and it could flank the ER membrane only once leaving the AP in cytoplasm. The construct can't re-enter ER as it lacks any more transmembrane domain.

construct 2 - the activity of alkaline phosphatase (AP) is high as the construct's N-terminal lies in ER lumen and it could flank the ER membrane only once leaving the AP in cytoplasm. The construct can't re-enter ER as it lacks any more transmembrane domain.

construct 3 - the activity of alkaline phosphatase (AP) is low as the construct's C-termianl as well as N-terrminal lies in ER lumen, bringing AP in lumen only. The construct can't move to cytoplasmic face as it lacks the third transmembrane domain.

construct 4 - the activity of alkaline phosphatase (AP) is low as the construct's C-termianl as well as N-terrminal lies in ER lumen, bringing AP in lumen only. The construct can't move to cytoplasmic face as it lacks the third transmembrane domain.

construct 5 - here AP has high activity because it lies on the cytoplasmic face. The protein flanks the membrane 3 times starting from ER, the last transmembrane domain brings AP to cytoplasm and hence it has high activity.

construct 6 - the whole protein is intact. The N-terminal of the protein lies in lumen and the C-terminal in cytoplasm so does AP. Hence it shows high AP activity.

D. removal of amino acids 55 to 85 will remove the transmembrane domain B leaving only two transmembrane domain to flank ER membrane (domain A and C). The new construct will flank ER membrane only two times, like the original protein the N-terminal will be in ER lumen while C-terminal along with AP will now wound up in ER lumen. Hence the activity of AP will decrease.

cytoplasm ER lumen