You are studying how proteins are targeted to the mitochondria. MIP1 is in the mitochondrial inner membrane, whereas MIP2 is targeted to the matrix. You are most interested in how proteins get into the inner membrane, so you focus on MIP1.

A) Analysis of a GFP-tagged MIP1 in a Tom 20 yeast mutant suggests that MIP1 targeting is Tom 20-dependent. Where would the GFP-tagged MIP1 be found in this mutant? Include your rationale in your answer.

B) Another scientist has already identified the matrix-targeting sequence at the N-terminus of MIP1. You think you have also found a stop-transfer sequence. How would you determine if the sequence you identified is NECESSARY for targeting to the membrane? You have the following tools (and you don't have to use all of them):

-GFP-tagged MIP1

-GFP-tagged MIP1 with the putative stop-transfer sequence mutated

-GFP-tagged MIP2

-GFP-tagged MIP2 with the putative stop-transfer sequence added

-Yeast

Describe the experiment including controls. State your predicted results if the sequence is necessary for inner membrane targeting.