Both Amino- and Carboxy-Terminal Portions Are Required for Insertion of Yeast Porin into the Outer Mitochondrial Membrane

Abstract

Yeast porin, the major outer mitochondrial membrane protein, is synthesized without a cleavable extension peptide and post-translationally inserted into the membrane. When inserted into the membrane, it acquires resistance to externally added trypsin. To locate the sequences responsible for membrane insertion and topogenesis in the primary structure of yeast porin, we constructed several deletion and chimeric mutants of the porin cDNA. These cDNAs were expressed in vitro and the products were assayed for capacity to be correctly inserted into isolated mitochondria. It was thus found that deletion of the segment spanning residues 37-98 did not appreciably impair the insertion competence and the inserted protein became resistant to trypsin. On the other hand, the porin mutant lacking the segment consisting of residues 17-98 did not acquire the trypsin resistance, though it could bind to mitochondria specifically. Deletion of the carboxy-terminal 62 amino acid residues also abolished the capacity to be correctly inserted into mitochondria. We conclude that information required for membrane insertion and intramembranous topogenesis of the porin molecule is stored not only in the amino-terminal region but also in the carboxy-terminal portion.

Role of the N‐ and C‐termini of porin in import into the outer membrane of Neurospora mitochondria

“…However, evidence is accumulating that a single stretch of amino acids does not suffice as a targeting signal, because point mutations in certain charged residues [23], as well as larger internal deletions [22,23], influence the efficiency of import. Thus, in contrast to proteins which span the membrane once, targeting signals in multi-topic mitochondrial proteins, such as porin, are not comprised of short segments of these polypeptides.…”

“…1). Deletion of residues 17 98 [22] or 9-156 [23] of yeast porin abrogates import. However, it could not be excluded that structural changes prevented assembly of the mutant proteins into a protease-resistant state in the membrane.…”

“…The import efficiency of the 283-residue yeast porin is decreased when one of two residues, Lys234 and Lys236, is mutated to a neutral or negatively charged amino acid [23], Additionally, deletion of the last 62 residues of yeast porin prevents its import [22]. Clearly, more precise deletions at both the N-and C-termini are required to define the roles of these regions in the import process.…”

Lysine Residues at Positions 234 and 236 in Yeast Porin Are Involved in Its Assembly into the Mitochondrial Outer Membrane

“…Others have suggested that regions more toward the N terminus than Lys-234 and Lys-236 may be important in the assembly of porin (3,13). The ability of two mutants in which tracts of amino acids had been deleted from the N-terminal portion of porin were tested for their ability to insert into the outer membrane.…”

“…It has been proposed that the N terminus of porin may be involved in sorting to the outer membrane (3), and deletion of the C-terminal 62 amino acids (i.e. residues 222-283) has been shown to prevent in vitro assembly of yeast porin (13). In the experiments reported here, the ability of various point mutations and deletion mutations of yeast mitochondrial porin to insert into the outer membrane of yeast mitochondria has been evaluated.…”