In an attempt to understand the mechanisms of sorting of mitochondrial inner membrane proteins, we have analyzed the import of subunit 9 of the mitochondrial F1F0‐ATPase (Su9) from Neurospora crassa, an integral inner membrane protein. A chimeric protein was used consisting of the presequence and the first transmembrane domain of Su9 fused to mouse dihydrofolate reductase (preSu9(1‐112)‐DHFR). This protein attains the correct topology across the inner membrane (Nout‐Cin) following import. The transmembrane domain becomes first completely imported into the matrix, where after processing of the presequence, it mediates membrane insertion and export of the N‐terminal tail. Import and export steps can be experimentally dissected into two distinct events. Translocation of the N‐terminal hydrophilic tail out of the matrix was blocked when the presequence was not processed, indicating an important role of the sequences and charges flanking the hydrophobic domain. Furthermore, export was supported by a delta pH and required matrix ATP hydrolysis. Thus the hydrophobic transmembrane domain operates as a membrane insertion signal and not as a stop‐transfer signal. Our findings suggest that several aspects of this sorting process have been conserved from their prokaryotic ancestors.

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...

Export of N-terminal tails of mitochondrial inner membrane proteins from the mitochondrial matrix is a membrane potential-dependent process, mediated by the Oxa1p translocation machinery. The hydrophilic segments of these membrane proteins, which undergo export, display a characteristic charge profile where intermembrane space-localized segments bear a net negative charge, whereas those remaining in the matrix have a net positive one. Using a model protein, preSu9(1-112)-dihydrofolate reductase (DHFR), which undergoes Oxa1p-mediated N-tail export, we demonstrate here that the net charge of N-and C-flanking regions of the transmembrane domain play a critical role in determining the orientation of the insertion process. The N-tail must bear a net negative charge to be exported to the intermembrane space. Furthermore, a net positive charge of the C-terminal region supports this N-tail export event. These data provide experimental evidence that protein export in mitochondria adheres to the "positive-inside" rule, described for secindependent sorting of membrane proteins in prokaryotes. We propose here that the importance of a charge profile reflects a need for specific protein-protein interactions to occur in the export reaction, presumably at the level of the Oxa1p export machinery.