Question

As a scientist, you hypothesize that a particular sequence of amino acids leads to localization in the inner mitochondria membrane. How could you test this using a non-mitochondrial cytosolic protein? Include the following components in your answer.

Hypothesis: Sequence X leads to localization in the inner mitochondrial membrane

Experimental Control:

Experimental Protein:

Method (s):

Result/Analysis:

Answer 1

The mitochondrial signal sequence is a 10-70 amino acid long peptide sequence that is found at the N-terminus of the newly synthesized protein. It is an established sequence. So, we can simply check the alignment of the given sequence with the conserved sequence and determine whether given sequence can serve as the mitochondrial target sequence. It is an in silico approach.

Invitro approach:
Hypothesis: The given sequence can cause mitochondrial localization.
Experimental control: A cytosolic protein tagged with GFP or a cytosolic protein tagged with the mutant signal sequence and GFP
Experimental protein: A cytosolic protein tagged with the given peptide sequence and GFP.
Method: Transfect/transform both the control and experimental proteins into a host system and observe for their localization. A cytosolic protein with GFP tag is expected to be found in the cytosol. If the given peptide sequences confer localization to the mitochondria, we can observe the GFP signal in the mitochondria from the cytosolic protein+ signal peptide + GFP construct.
Results: If the peptide sequence confers localization to the mitochondria, we can find the GFP signal in the mitochondria.