Sorting of d-Lactate Dehydrogenase to the Inner Membrane of Mitochondria

Abstract

Nuclear encoded proteins of the inner membrane of mitochondria contain topogenic signals which function to sort them to the membrane following their import. These topogenic signals comprise hydrophobic cores of varying length and are flanked usually by charged amino acids. In many instances these signals form integral parts, the transmembrane anchors, of the sorted protein. In a small number of cases they are proteolytically cleaved following the sorting event. These topogenic signals are distinct from mitochondrial targeting sequences that serve to target the precursor initially to the mitochondria and to initiate membrane potential (⌬)-dependent translocation across the inner membrane. In some cases these topogenic signals are located after N-terminal mitochondrial targeting signals (presequences) and operate in conjunction with them. This is not the case for all topogenic signals as many proteins of the inner membrane are synthesized without presequences but rather contain internal mitochondrial targeting signals.Bearing these differences in mind, together with the wide variety of orientations displayed by inner membrane proteins, it would appear unlikely that the topogenic signals operate in an uniform manner. How do they act to ensure the sorting of proteins to the inner membrane? Recently we have described that the topogenic signals of a subset of inner membrane proteins serve as export signals directing the export of domains of the protein from the matrix to the intermembrane space following the complete import of the preprotein into the matrix (1-3). On the other hand, it has been suggested that some topogenic signals function as translocation arrest signals at the level of the inner membrane, thereby preventing further import into the matrix (4 -8).To The data presented here demonstrate that D-LD is a mitochondrial protein anchored to the inner membrane with an N in -C out orientation. We present here information on the topogenic signal sequence and energetic requirements necessary for D-LD to achieve this membrane orientation. EXPERIMENTAL PROCEDURESIsolation of Yeast Mitochondria-S. cerevisiae wild-type strain (D273-10B) was grown in lactate medium (10) at 30°C, while the temperature-sensitive mutant of mt-Hsp70, ssc1-3 (PK83) and its corresponding wild type (PK82) (11) were grown at 24°C. Cells were harvested at an A 578 of ϳ1, and mitochondria were isolated, as described previously (10), with the exception that the zymolyase treatment of PK82 and PK83 strains was performed at 24°C. Isolated mitochondria were resuspended in 0.6 M sorbitol, 20 mM HEPES, pH 7.4, 1 mM EDTA (SEH-buffer) at a protein concentration of 10 mg/ml.Recombinant DNA Techniques and Plasmid Constructions-The recombinant DNA techniques applied were as described by Sambrook et al. (12). The D-LD gene was obtained by amplification of yeast genomic DNA of strain D273-10B by a polymerase chain reaction. The resulting DNA fragment was cloned into pGEM4 (Promega, Madison, WI) yielding the plasmid pDLD. To construct the plasmids for the e...

Processing of Mgm1 by the Rhomboid-type Protease Pcp1 Is Required for Maintenance of Mitochondrial Morphology and of Mitochondrial DNA

“…A similar behavior upon alkaline treatment of mitochondria was observed for D-lactate dehydrogenase (Fig. 2B), a protein that is anchored to the inner membrane by a single N-terminal transmembrane segment and faces the intermembrane space (29). In contrast, the ADP/ ATP carrier (Aac2), an integral membrane protein with six membrane-spanning segments (27), was completely resistant to extraction by carbonate (Fig.…”

Molecular characterization of Saccharomyces cerevisiae Sco2p reveals a high degree of redundancy with Sco1p

“…Such an arrangement is present in a variety of mt inner membrane proteins. In the case of D-lactate dehydrogenase, which attains a similar N in -C out orientation in the inner membrane, it was shown that the TM segment and the following cluster of charged aa act as a bipartite sorting signal (Rojo et al, 1998).…”

Charged flanking residues control the efficiency of membrane insertion of the first transmembrane segment in yeast mitochondrial Mgm1p

“…Previous studies [10,16,17] have shown that the presence of charged residues in the neighborhood of a transmembrane domain can affect membrane integration in the mitochondrial inner membrane. Consistent with this, statistical analysis on the distribution of charged residues flanking the transmembrane domains of mitochondrial inner membrane proteins that follow the 'stop-transfer' pathway has shown that positively charged Lys and Arg residues are abundant on both sides of the TM segments, whereas negatively Fig.…”