Biogenesis of yeast dicarboxylate carrier: the carrier signature facilitates translocation across the mitochondrial outer membrane.
A family of related carrier proteins mediates the exchange of metabolites across the mitochondrial inner membrane. The carrier signature Px[D/E]xx[K/R] is a highly conserved sequence motif in all members of this family. To determine its function in the biogenesis of carrier proteins, we used the dicarboxylate carrier (DIC) of yeast as a model protein. We found that the carrier signature was dispensable in binding of the newly synthesized protein to the import receptor Tom70, but that it was specifically required for efficient translocation across the mitochondrial outer membrane. To determine the relevance of individual amino acid residues of the carrier signature in the transport activity of the protein, we exchanged defined residues with alanine and reconstituted the mutant proteins in vitro. Substitution of the carrier signature in helix H1 reduced the transport activity for [(33)P]-phosphate by approximately 90% and an additional substitution of the carrier signature in helix H5 blocked the transport activity completely. We conclude that the carrier signature of the dicarboxylate carrier is involved both in the biogenesis and in the transport activity of the functional protein.
Zara
Vincenzo
V
Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Provinciale Lecce-Monteroni, I-73100 Lecce, Italy. vincenzo.zara@unile.it
Ferramosca
Alessandra
A
Capobianco
Loredana
L
Baltz
Katrin M
KM
Randel
Olga
O
Rassow
Joachim
J
Palmieri
Ferdinando
F
Papatheodorou
Panagiotis
P
D000409:Alanine / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D029382:Dicarboxylic Acid Transporters / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013462:Sulfur Radioisotopes / Q000378:metabolism; D013997:Time Factors
D029382:Dicarboxylic Acid Transporters; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D029701:Saccharomyces cerevisiae Proteins; D013462:Sulfur Radioisotopes; D000409:Alanine