An amino-terminal signal sequence abrogates the intrinsic membrane-targeting information of mitochondrial uncoupling protein.

Mitochondrial uncoupling protein, a polytopic integral protein of the inner membrane, is initially made in the cytoplasm as a soluble polypeptide (307 amino acids) lacking a cleavable targeting (signal) peptide. Earlier studies (Liu, X., Bell, A. W., Freeman, K. B., and Shore, G. C. (1988) J. Cell Biol. 107, 503-509) identified internal regions of the molecule that are critical for targeting and membrane insertion. Here, we demonstrate that the ability of uncoupling protein to insert into the inner membrane is abrogated when the molecule is fused behind the matrix-targeting signal of preornithine carbamyltransferase; the hybrid protein was imported across the inner membrane and deposited in the matrix where it was processed. In this context, however, the processed product remained in the matrix and was incapable of inserting into the inner membrane.

Here, we demonstrate that the ability of uncoupling protein to insert into the inner membrane is abrogated when the molecule is fused behind the matrix-targeting signal of preornithine carbamyltransferase; the hybrid protein was imported across the inner membrane and deposited in the matrix where it was processed. In this context, however, the processed product remained in the matrix and was incapable of inserting into the inner membrane.
Uncoupling protein (UCP)' is an integral protein of the mitochondrial inner membrane in brown adipose tissue (1). It shares strong structural similarities with two other proteins of the inner membrane, ADP/ATP carrier (AAC) and phosphate carrier (2,3). All three consist of a 3-fold repeat of -100 amino acids, with each repeat predicted to consist of a pair of transmembrane segments connecting an ectodomain, which, in the case of AAC and UCP, is exposed to the matrix (2)(3)(4). The transmembrane segments exhibit amphiphilic characteristics and, therefore, are probably stabilized in the membrane as paired helical structures (2).
Analysis of UCP (5)  tide, one located within the NHp-terminal third of the molecule (i.e. within the first repeat domain) and the other downstream of this position. Neither protein is made as a higher molecular weight precursor (2,5,9,10). In the case of UCP, the amino-terminal third is responsible for both targeting and membrane insertion (5) and as well may be required to help mediate insertion of the rest of the molecule (5). We recently suggested that UCP follows a coordinate insertion pathway during import into mitochondria in which the three pairs of membrane-spanning segments are threaded into the inner membrane led by matrix-targeting signals located in the ectodomains (5). Whether insertion occurs during unidirectional translocation of UCP across the inner membrane or whether UCP follows a "conservative" sorting pathway in which the molecule is translocated first to the soluble matrix compartment and then back into the inner membrane (10) is not presently known.
Here, we have examined the fate of a hybrid molecule in which the entire 3-fold repeat structure of UCP, containing all of the requisite topogenic information for mitochondrial targeting and membrane insertion, is placed behind a matrixtargeting signal derived from the precursor to the matrix enzyme, ornithine carbamyltransferase. The hybrid protein was imported to the matrix compartment where the preornithine carbamyltransferase signal sequence was removed. The resulting UCP molecule remained in the matrix rather than inserting into the inner membrane.

EXPERIMENTAL PROCEDURES
General-Routine procedures for recombinant DNA manipulation, transcription in the pSP64 system, translation in a rabbit reticulocyte lysate in the presence of [%]methionine, isolation of rat heart mitochondria, in vitro import into mitochondria, and analysis of import products by SDS-PAGE and fluorography were described previously (5,11,12

AND DISCUSSION
Transcription-translation of UCP cDNA in a pSP64 vector yields two products. Analysis of these products by SDS-PAGE is consistent with translation initiation occurring at UCP codons 1 and 13 (5). Both products, designated UCP and UCPdl.12, respectively, are imported to the inner membrane of rat heart mitochondria in vitro, as judged by their acquisition of A$-dependent resistance to both exogenous protease and extraction at pH 11.5 (5). UCP is not made as a larger Inner Membrane Uncoupling Protein precursor molecule so that the requirement of an electrochemical potential for membrane insertion is necessary to distinguish between product inserted into the inner membrane (A+dependent) and product adventitiously and perhaps cryptically associated with the surface of the organelle (A#-independent) (5). As illustrated previously, the deletion mutant UCPd,.,.' is imported and inserted into the inner membrane indicating that UCP amino acids 1-12 are dispensable for both targeting and membrane anchoring. Import ofpO-UCP-cDNA encoding UCP was inserted into the pSP64 vector and was then modified to include a cDNA fragment encoding the first 37 amino acids of preornithine carbamyltransferase fused to UCP amino acids 10-307; as well, the ATG codon at UCP amino acid position 13 was deleted to avoid internal initiation of translation at this position. Thus, removal of the preornithine carbamyltransferase signal sequence (32 amino acids) from the hybrid protein would yield a "mature" product in which 5 amino acids of mature processed preornithine carbamyltransferase (SQVQL) replace 9 amino acids (MVSSTTSEV) from the NHs-terminus of UCP. The intrinsic targeting information of UCP, however, exists downstream of amino acid 12 (5). Synthesis of PO-UCP in a rabbit reticulocyte lysate in vitro yielded a single polypeptide product with an expected size of -37 kDa (Fig. 1, lane 1). The hybrid precursor polypeptide was imported and processed by isolated mitochondria (Fig. 1, lane 4) in a manner that was dependent on an electrochemical potential across the inner membrane ( Fig. 1, lanes 2 and 3); precursor on the surface of the organelle was sensitive to exogenous protease, whereas the processed product was protected ( Fig. 1, lanes 3 and 5). The extent and characteristics of PO-UCP import into mitochondria were very similar to that observed for UCPdl.ls, except of course that processing PO-UCP UC& I-I 2 of UCPaI.,r to a smaller product did not take place (Fig. 1,  lanes 6-10). Because PO-UCP was processed to the expected size following import into mitochondria in uitro, its NH*-terminal preornithine carbamyltransferase signal sequence presumably gained access to the matrix compartment where the Zn'+dependent preornithine carbamyltransferase processing enzyme is located (14). As expected, therefore, import of PO-UCP in the presence of o-phenanthroline, a Zn2+ chelator, resulted in partial inhibition of precursor processing, with the result that the accumulated precursor acquired resistance to exogenous trypsin (Fig. 2, lane 2); this is in contrast to import in the absence of chelator in which all of the full-size precursor that cosedimented with mitochondria was sensitive to the protease (Fig. 1, lanes 3 and 5). Localization of Precursor and Processed PO-UCP-An initial examination of processed PO-UCP inside mitochondria revealed that it was not integrated into a lipid bilayer and remained completely extractable by 0.1 M Na2C03, pH 11.5 (not shown). This analysis was extended to include the fullsize precursor that was allowed to accumulate in a proteaseresistant compartment following import in the presence of ophenanthroline (Fig. 2, lane 2); it too was extracted by alkali (Fig. 2, lane 3). When the inhibition of processing was relieved by the addition of excess Zn'+, most of the precursor was processed (Fig. 2, lane 4), and the resulting processed product was released by alkaline treatment (Fig. 2,lane 5). This is in contrast to imported UCPdl.12 which was resistant to alkaline extraction ( Fig. 2, lanes 7 and 8). Following import, mitochondria were treated with trypsin to remove UCP adhering to the surface of the organelle (Fig. 1) and were then extracted with 0.1 M Na&O:s, pH 11.5, and the membranes recovered by high speed centrifugation (Fig. 2). The percent recovery of imported UCPdl.12 after protease, then alkaline, treatment was similar to the recovery of an endogenous marker for integral proteins of the inner membrane of heart mitochondria, ADP/ ATP carrier protein (not shown).
The fact that import of PO-UCP was dependent on an ++++ --0-PHE. electrochemical gradient across the mitochondrial inner membrane (Fig. l), that processing was carried out by the Zn*'dependent signal peptidase (Fig. 2), and that the resulting processed product was released by alkaline extraction (Fig. 2) suggests that it accumulated in the soluble matrix compartment rather than being retained in the inner membrane. This conclusion was extended by the results presented in Fig. 3, which rule out the possibility that processed PO-UCP was located in the intermembrane space; rather, it co-localized with processed preornithine carbamyltransferase, a matrix marker. Following import of PO-UCP or preornithine carbamyltransferase, mitochondria were treated with various concentrations of digitonin to disrupt the outer membrane, and the sensitivities of processed preornithine carbamyltransferase, processed PO-UCP, and PO-UCP (which was allowed to accumulate inside mitochondria in the presence of o-phenanthroline) to exogenous trypsin were determined (Fig. 3). All three polypeptides were equally resistant to trypsin, up to a concentration of 0.75 mg of digitonin/mg of mitochondrial protein; in contrast, disruption of the outer membrane by 0.5-0.75 mg of digitonin resulted in the loss of latency of succinate cytochrome c oxidoreductase activity, as indicated by exogenous substrate (cytochrome c) gaining access to the enzyme which is located on the outer aspect of the inner membrane (13). The partial sensitivity to trypsin of the three polypeptides that was observed at 0.75 mg of digitonin presumably  reflected partial damage to the inner membrane; by 1.0 mg of digitonin, complete sensitivity to trypsin of the three polypeptides was observed as well as a net loss in total reductase activity (Fig. 3), indicating that the inner membrane was severely damaged, resulting in accessibility of the matrix compartment to the protease. Finally, Ekerskorn and Klingenberg (15) have recently observed that native UCP in brown adipose mitochondria yields two fragments upon partial digestion by trypsin of mitochondria that were frozen and thawed to disrupt the outer membrane. The major fragment (Ti) was -30 kDa in size and was generated by removal of -2 kDa from the COOHterminus of UCP; a minor fragment (T2) of -25 kDa arose with a time delay from T1 (15). As shown in Fig. 4, fragments similar to those demonstrated in Ref. 15 (Ti and T2) were obtained when rat heart mitochondria containing newly imported UCPdl-ip were treated under similar conditions; treatment of frozen-thawed mitochondria with trypsin for progressively longer times led to the disappearance of full-size UCPdl-i2 and the appearance of Ti and TP (Fig. 4, lanes l-6). This is in contrast to the situation for processed PO-UCP which remained largely intact following trypsin treatment of frozen-thawed mitochondria (Fig. 4, lanes 7-12). These findings ( Fig. 4) are consistent with the conclusion that UCPdi.ip was integrated into the inner membrane in a disposition similar to native UCP, while processed PO-UCP resided in the matrix. The minor band that appeared immediately below processed PO-UCP in mitochondria that had been treated with trypsin for 60-90 min (Fig. 4, lanes 11 and 12) was also evident in certain incubations that had not received trypsin treatment (e.g. see Fig. 1, lane 4) and, therefore, might result from the action of endogenous proteases over the extended period of these incubations.

CONCLUDING REMARKS
The polytopic disposition of UCP in the inner membrane of mitochondria is determined by multiple topogenic signals which are located within amino acids 12-105 as well as downstream of this position (5). Here, we have demonstrated that the membrane-anchoring function of UCP, which results in the assembly of 3 pairs of amphiphilic cy-helices spanning the inner membrane (2,3), is abrogated when UCP amino acids lo-307 are fused behind a matrix-targeting signal derived from preornithine carbamyltransferase. In this context, the fusion protein is translocated to the soluble matrix compart-Inner Membrane Uncoupling Protein ment of mitochondria in vitro where it is processed by the Zn2+-dependent matrix-processing enzyme. Presumably, the presence of the preornithine carbamyltransferase signal sequence confers a conformation to the rest of the molecule which is no longer compatible with insertion into the inner membrane, i.e. via paired amphiphilic helices led by internal matrix-targeting domains (5). It may also be that UCP and PO-UCP employ different receptors for import (10) and that only the UCP receptor is capable of presenting the protein to the inner membrane in a form competent for insertion. Interestingly, however, insertion into the inner membrane is abrogated even after the preornithine carbamyltransferase signal sequence has been removed in the matrix. Either an insertion-incompetent conformation is retained in the processed form of the protein or the machinery requisite for protein integration into the inner membrane is not available to the processed product when the protein is presented to the 3.