Question

you have shown that protein Abc1 (encoded by the ABC1gene) is a transcriptional activator of genes that have the upstream activation sequence of UASABC in their promoters. preliminary experiments utilizing cells expressing a fusion protein that you have constructed in which Abc1 is fused to GFP suggest that the protein has dual localization in the ER and the nucleus. you hypothesize that: a) Abc1 is tethered to the ER during repressing conditions and translocates to the nucleus during derepressing conditions; b)Abc1 bind to the phospholipid phosphatidic acid (PA); and c) binding to PA is essential for maintaining Abc1 in the ER during repressing conditions. Propose experiments to definitively test your hypothesis. for the purpose of the question, assume that you cannot manipulate cellular PA levels.

Answer 1

a) I'll clone this activation sequence (UASABC) of Abc1 in front of a reporter (luciferase) gene. I'll coexpress this reporter construct alongwith fused GFP-Abc1. I'll screen for derepressing conditions, of the conditions are not known. The increase in reporter activity occurs only when conditions are derepressing or activating. Then I'll check localization of GFP in both repressing and derepressing conditions. In repressing conditions, ER associated GFP-Abc1 will be more and in derepressing conditions nuclear GFP-Abc1 will be more.

b) I'll do a lipid-protein overlay assay for checking the binding activity of Abc1 to phosphatidic acid (PA). Here, PA will be coated onto a PVDF membrane. If antibody for Abc1 is available I'll directly use recombinant Abc1. If antibody is not available, I'll tag Abc1 with His or GST or GFP, I'll purify recombinant protein and use that. Purified protein will be added to membrane containing PA, washed extensively and will be developed using primary and enzyme linked secondary antibodies. This confirms whether Abc1 binds to PA or not.

For confirming in vivo, I'll check the sequence of Abc1 for the similarities with previously known PA binding domains. If found those domains will be used directly, if not found deletion mutants will be generated to identify the region resposnsible for PA binding.

c) Once the binding region was found, the PA binding deficient Abc1 (tagged with GFP) will be overexpressed and checked under repressing conditions. Both nuclear localization and increase in reporter activity should happen, as this cannot bind to PA for repressing to happen. If repressing still happens, the mode of repression may not be through PA.