Question

Compare and contrast protein import into the mitochondria and nucleus. Include the following terms in the way that shows you understand differences in the import mechanisms: (1) translocation mechanism, (2) signal sequences (whether the signal sequence is cleaved following import) and (3) state of protein folding (whether the proteins are in an unfolded or folded state during the import).

Answer 1

Matrix proteins must pass from cytosolic polyribosomes through the outer and inner mitochondrial membranes to reach
their destination. Passage through the two membranes is called translocation. They have an amino terminal leader sequence (pre-sequence), about 20 to 50 amino acids in length which is not highly conserved but is amphipathic and contains many hydrophobic and positively charged amino acids (eg, Lys or Arg). The presequence is equivalent to a signal peptide mediating attachment of polyribosomes to membranes of the ER, but in this instance targeting proteins to the matrix.Translocation occurs posttranslationally, after the matrix proteins are released from the cytosolic polyribosomes. Interactions with a number of cytosolic proteins that act as chaperones and as targeting factors occur prior to translocation. Two distinct translocation complexes are situated in the outer and inner mitochondrial membranes, referred to (respectively) as TOM (translocase-of-the-outer membrane) and TIM (translocase-of-the-inner membrane). Each complex has been analyzed and found to be composed of a number of proteins, some of which act as receptors (eg, Tom20/22 ) for the incoming proteins and others as components (eg, Tom40 ) of the transmembrane pores through which these proteins must pass. Proteins must be in the unfolded state to pass through the complexes, and this is made possible by ATP-dependent binding to several chaperone proteins includ-ing Hsp70. In mitochondria, chaperones are involved in translocation, sorting, folding, assembly, and degradation of imported proteins. A proton-motive force across the inner membrane is required for import; it is made up of the electric potential across the membrane (inside negative) and the pH gradient. The positively charged leader sequence may be helped through the membrane by the negative charge in the matrix. The presequence is split off in the matrix by a matrix-processing protease (MPP). Contact with other chaperones present in the matrix is essential to complete the overall process of import. Interaction with mt-Hsp70 (mt = mitochondrial; Hsp = heat shock protein; 70 =∼70 kDa) ensures proper import into the matrix and prevents misfolding or aggregation, while interaction with the mt-Hsp60–Hsp10 system ensures proper folding. The interactions of imported proteins with the above chaperones require hydrolysis of ATP to drive them.

The general picture that has emerged is that proteins to be imported (cargo molecules) carry a nuclear localization signal (NLS). One example of an NLS is the amino acid sequence (Pro)2-(Lys)3-Arg-Lys-Val, which is markedly rich in basic residues. Depending on which NLS it contains, a cargo molecule interacts with one of a family of soluble proteins called importins, and the complex dockstransiently at the NPC. Another family of proteins called Ran plays a critical regulatory role in the interaction of the complex with the NPC and in its translocation through the NPC. Ran proteins are small monomeric nuclear GTPases and, like other GTPases, exist in either GTP-bound or GDP-bound states. They are themselves regulated by guanine nucleotide exchange factors (GEFs), which are located in the nucleus, and Ran GTPase-accelerating proteins (GAPs), which are predominantly cytoplasmic. The GTP-bound state of Ran is favored in the nucleus and the GDP-bound state in the cytoplasm. The conformations and activities of Ran molecules
vary depending on whether GTP or GDP is bound to them (the GTP-bound state is active). The asymmetry between nucleus and cytoplasm—with respect to which of these two nucleotides is bound to Ran molecules—is thought to be crucial in understanding the roles of Ran in transferring complexes unidirectionally across the NPC. When cargo molecules are released inside the nucleus, the importins recirculate to the cytoplasm to be used again.

Mitochondria -

a. Translocation mechanism - explained

b. Signal sequence cleaved following import

c. Proteins are in unfolded state.

Nucleus -

a. Translocation mechanism - explained

b. Signal sequence is not cleaved following import

c. Proteins are in folded state.