To determine how the protein is arranged in the ER membrane, you use a series of

Question

To determine how the protein is arranged in the ER membrane, you use a series of truncations that are connected at the C-terminus to another reported protein, alkaline phosphatase (AP, blue star). Your constructs are shown in the following Figure, where the yellow bar shows the amino acids of 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 is located in the ER lumen. Draw the structure of Construct 6 in the following diagrams of the ER membrane. Your drawing should contain boxes with letters and wave lines to represent YFG1 and a star to represent AP. in Part A, add the flanking charges around transmembrane domain A that would lead to your predicted protein orientation. Please explain how the protein structures of Constructs 1-6 in the ER membrane lead to the observed activities of alkaline phosphatase. in a follow-up experiment, your lake 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.

Explanation / Answer

A) cytoplasm -GFP -ER fusion protein (GFP -Sec617) expressed in a COS cell

ER membrane -targetting and retention of inner nuclear membrane (INM) Proteins.the newly translated protein is translocated into ER membrane .

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