Question

Describe the main mechanisms by which newly synthesized proteins are transported from the cytosol to mitochondria and outline the importance of signal sequences and protein processing in the localization of proteins within the different compartments of the mitochondrion.

Answer 1

Main mechanism of transport of mitochondrial proteins from cytosol

All the cellular proteins are synthesized in the cytosol by the rough endoplasmic reticulum (RER) where the ribosomes take part in the translation of mRNA with the help of tRNA that bring the corresponding amino acids to build the polypeptide chain in correspondence to the triplet codons in mRNA sequence. Mitochondrial precursor proteins coded by nuclear DNA ,are synthesized fully before they get transported to the mitochondria from cytosol as opposed to co-translocation of proteins in ER. Several post-translation events aid the process of transport.

Mitochondria can be divided into two compartments due to the presence of an inner membrane that form cristae around the mitochondrial matrix and an outer membrane that gives the closure and form intermembrane space. Most of the mitochondrial precursor proteins contain a signal sequence that is cleaved by the signal peptidase upon delivery. Research shows that the protein structure is also important in this transport mechanism. The precursor proteins have hugh propensity of forming an amphipathic alpha helix. The positively charged residues are clustered at one side of the helix and uncharged hydrophobic residues cluster at the opposite side of the helix . This secondary conformation can be identified by the proteins that initiate the translocation process. Cytosolic proteins of family hsp70 bind to these precursor unfolded polypeptides in order to prevent unfolded protein response within cell. The subsequent release of the protein from hsp70 proteins require ATP hydrolysis.

There are two translocase complexes in the outer membrane and the inner membrane called TOM and TIM respectively. The N-terminal signal sequence of precursor protein is recognized by the TOM complex and the TOM complex translocates to the membrane contact sites and insert the protein along with the signal sequence in the intermembrane space. The TIM23 complex then transfer some of the proteins to matrix and helps to insert transmembrane proteins to the inner membrane. The TIM22 complex mediates the insertion of a subclass of inner membrane proteins along with the carrier proteins. Oxa, a third protein translocator at the inner membrane, mediates the insertion of inner membrane proteins that are synthesized within the mitochondria. The translocation of precursor proteins by TIM complex require a gradient of H⁺ ions which is created by the pumping of H⁺ ions from mitochondrial matrix to the inner membrane space.

Once the unfolded protein emerges from the translocator to the matrix, mitochondrial hsp70 proteins bind it and releases subsequently in an ATP dependent manner. This gives the translocation a specific direction and prevent backsliding. Once free from the hsp70, unfolded proteins are bound by mitochondrial hsp60 proteins that help them to fold properly, deriving the necessary energy from ATP hydrolysis.

Importance of Signal sequences and protein processing in compartment specific localization of proteins

1. The innermembrane proteins or the proteins destined to be in the intermembrane space are mostly transported to the mitochondrial matrix by the TOM-TIM complexes. Once in the matrix , the N-terminal signal sequence is chopped off by the matrix signal peptidase. However, these proteins contain a hydrophobic amino acid sequence right next to the signal sequence that work as a new signal sequence for their transport to the inner membrane or intermembrane space by the Oxa complex.

2. Another mechanism is there which prevents the translocation of proteins to the matrix. The N terminal sequence is translocated by TIM complex in the inner membrane. A second sequence following the original signal sequence works as a stop-transfer signal and stalls the translocation. Inside the matrix the N-terminal signal sequence is cleaved by the signal peptidase and the rest of the polypeptide is pulled by the TOM complex and placed in the intermembrane space. The second hydrophobic stop-transfer sequence is cleaved by the signal peptidase in the intermembrane space and the rest of the polypeptide is retained as soluble protein of intermembrane space.