Question

You hypothesize that a protein is localized to the mitochondria in a cell.

A. Design a microscopy experiment to test this hypothesis. Please include the labels and types of microscopy you would use.

B. How might you test this hypothesis without using a microscopy technique?

Answer 1

B.)

Most mitochondrial proteins are synthesized as cytosolic precursors containing uptake peptide signals. Cytosolic chaperones deliver preproteins to channel linked receptors in the mitochondrial membrane. The preprotein with presequence targeted for the mitochondria is bound by receptors and the General Import Pore (GIP) (Receptors and GIP are collectively known as Translocase of Outer Membrane or TOM) at the outer membrane. The preprotein is translocated through TOM as hairpin loops. The preprotein is transported through the intermembrane space by small TIMs (which also acts as molecular chaperones) to the TIM23 or 22 (Translocase of Inner Membrane) at the inner membrane. Within the matrixthe targeting sequence is cleaved off by mtHsp70.

Three mitochondrial outer membrane receptors are known: TOM20, TOM22 and TOM70
TOM70: Binds to internal targeting peptides and acts as a docking point for cytosolic chaperones.
TOM20: Binds presequences
TOM22: Binds both presequences and internal targeting peptides
The TOM channel (TOM40) is a cationspecific high conductance channel with a molecular weight of 410 kDa and a pore diameter of 21Å.

The presequence translocase23 (TIM23) is localized to the mitochondrial inner membrane and acts a pore forming protein which binds precursor proteins with its N-terminus. TIM23 acts a translocator for preproteins for the mitochondrial matrix, the inner mitochondrial membrane as well as for the intermembrane space. TIM50 is bound to TIM23 at the inner mitochondrial side and found to bind presequences. TIM44 is bound on the matrix side and found binding to mtHsp70.
The presequence translocase22 (TIM22) binds preproteins exclusively bound for the inner mitochondrial membrane.

Mitochondrial matrix targeting sequences are rich in positively charged amino acids and hydroxylated ones.

Proteins are targeted to submitochondrial compartments by multiple signals and several pathways.

Targeting to the outer membrane, intermembrane space, and inner membrane often requires another signal sequence in addition to the matrix targeting sequence. Thus through amino acid sequence we can analyse wether the protein is localised in a mitochondrion or not