Abstract
Tom70p is targeted and inserted into the mitochondrial outer membrane in the Nin-Ccyto orientation, via an NH2-terminal signal anchor sequence. The signal anchor is comprised of two domains: an NH2-terminal hydrophilic region which is positively charged (amino acids 1-10) followed by the predicted transmembrane segment (amino acids 11-29). Substitution of the NH2-terminal domain with a matrix-targeting signal caused the signal anchor to adopt the reverse orientation in the outer membrane (Ncyto-Cin) or, if presented to mitoplasts, to arrest protein translocation at the inner membrane without insertion. Physically separating the transmembrane segment from the matrix-targeting signal by moving it downstream within the protein resulted in a failure to arrest in either membrane, and consequently the protein was imported to the matrix. However, if the mean hydrophobicity of the Tom70p transmembrane segment was increased in these constructs, the protein inserted into the inner membrane with an Nin-Cout orientation. Therefore we have determined conditions that allow the Tom70p transmembrane domain to insert in either membrane, pass through both membranes, or arrest without insertion in the inner membrane. These results identify the mean hydrophobicity of potential transmembrane domains within bitopic proteins as an important determinant for insertion into the mitochondrial inner membrane.
Highlights
Nuclear-encoded precursor proteins destined for import into mitochondria are sorted to one of four compartments in the organelle: outer or inner membrane, intermembrane space, or matrix
Replacement of the extreme hydrophilic NH2 terminus of the pOMD29 signal anchor with the matrix-targeting signal of pre-ornithine carbamoyltransferase (pOCT) created pO-SA 36, which inserts into the outer membrane in an orientation opposite that of pOMD29, i.e. Ncyto-Cin (Fig. 5A) [17, 20]
Deletion of the predicted transmembrane portion of the pOMD29 signal anchor abolishes the ability of the protein to target mammalian mitochondria in vitro [4], whereas the pO-SA 36 fusion construct containing the pOCT matrix-targeting signal but lacking the Tom70p transmembrane segment is efficiently imported to the matrix [34, 35] (Fig. 5A)
Summary
Nuclear-encoded precursor proteins destined for import into mitochondria are sorted to one of four compartments in the organelle: outer or inner membrane, intermembrane space, or matrix. Stop-transfer sequences do not contain intrinsic membraneselective targeting information but rather they are passive transmembrane segments that are located downstream of matrix-targeting signals, causing an otherwise matrix-destined protein to arrest translocation and insert into the outer or inner membrane. Another model proposes that following removal of the NH2 terminus in the matrix, the hydrophobic domain redirects the precursor protein back from the matrix compartment to the intermembrane space [31] It has been consistently observed, that the hydrophobic domain, while capable of translocation arrest within the inner membrane import machinery, does not integrate into the surrounding bilayer. This domain is functionally different from the structurally analogous regions in signal anchor and stop-transfer sequences
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