©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lysine Residues at Positions 234 and 236 in Yeast Porin Are Involved in Its Assembly into the Mitochondrial Outer Membrane (*)

(Received for publication, June 15, 1995; and in revised form, September 20, 1995)

Mitchell D. Smith (1) Michelle Petrak (1) Paul D. Boucher (1) Kenneth N. Barton (1) Latisha Carter (1) Gowri Reddy (1) Elizabeth Blachly-Dyson (2) Michael Forte (2) Jeannie Price (3) Keith Verner (3) Roy B. McCauley (1)(§)

From the  (1)Department of Pharmacology, School of Medicine, Wayne State University, Detroit, Michigan 48201, the (2)Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201, and the (3)Department of Cellular and Molecular Physiology, Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Various point mutations of lysyl residues in yeast mitochondrial porin (283 residues) were tested for their ability to assemble in vitro into the outer membranes of intact yeast mitochondria. Assembly was evaluated by protection from proteinases. The extent of assembly of two of the mutants, K234E and K236E porins, was much less than for wild-type in either post-translational or co-translational assembly assays. Lysine to glutamate mutants at other positions and K234R porin assembled as well as wild-type, but K234Q porin was poorly inserted. When both Lys-234 and Lys-236 were mutated, K234R/K236R porin was inserted better than K234Q/K236Q porin, which was inserted better than K234E/K236E; however, none of these mutants assembled as well as wild-type porin. It was concluded that optimal assembly of yeast porin depended on the presence of positively charged residues at both positions 234 and 236 and a lysine at one of these positions. After undergoing the assembly reaction, mutants that were vulnerable to proteinase K (i.e. K234E, K234Q, and K236E porins) seemed to be incompletely digested and were, to varying degrees, resistant to extraction by Na(2)CO(3) (pH 11.5). These experiments suggested that these mutants were incompletely inserted into the outer membrane. Both Lys-234 and Lys-236 are included in an internal pentapeptide, VKAKV, that is conserved in porins from protists, plants, and animals, and it is possible that, at least, the lysines in this tract are one of the signals for the membrane assembly of these proteins.


INTRODUCTION

Mitochondrial porin (i.e. the 283-residue mitochondrial outer membrane voltage dependent anion channel) forms large holes in the mitochondrial outer membrane that permit the passage of metabolites necessary for reactions that occur in the interior compartments of mitochondria (for review, see (1) ). It is likely that each channel is formed by a single M(r) 31,000 porin polypeptide(2) . Although the tertiary structure of eukaryotic porin is still unknown, it is thought to be largely comprised of amphipathic beta-strands in the form of a beta-barrel. This conclusion is based in part on observations that fungal porin does not contain sufficiently long hydrophobic tracts to serve as membrane anchors (3) and in part, in analogy with the known structures of bacterial porins(4, 5) . The distribution of amino acid residues that contribute to the channel properties of porin has been interpreted to mean that yeast porin has 13 membrane spanning tracts: a single amphipathic alpha-helix and 12 amphipathic beta-strands(6) . According to this model, charged amino acids in the membrane spanning tracts line the aqueous pore.

Mitochondrial porins are encoded by nuclear genes and synthesized by cytosolic ribosomes. Porin has been shown to assemble in vitro by both post-translational (7, 8, 9) and co-translational processes(10) . Porin assembly is independent of the mitochondrial membrane potential but it seems to depend on energy in the form of ATP (3, 9, 11) . As is the case for other outer membrane proteins, the mature (i.e. membrane-assembled) form of porin is the same size as its precursor, suggesting that tracts of amino acids that may be involved in the sorting of porin to the outer membrane and its insertion into the membrane are retained in the mature protein. Although it seems clear that N-terminal or C-terminal tracts are crucial for the assembly of bitopic outer membrane proteins(12) , much less is known about porin. 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. These experiments indicated that Lys-234 and Lys-236 were involved in porin assembly. Substitution of two other polar amino acids, glutamate and glutamine, impaired assembly. Optimal assembly occurred when both residues at positions 234 and 236 were positively charged and lysine was present at one of these positions. Lys-234 and Lys-236 exist in an internal pentapeptide (VKAKV) in yeast porin that is conserved in 10 other porins from protists, plants, and mammals. It is suggested that, at least, in yeast porin, the basic residues in this tract may have an important role in the membrane insertion of this protein.


EXPERIMENTAL PROCEDURES

The methods for the preparation of mitochondria from Saccharomyces cerevisiae, RNA transcription with the SP6 RNA polymerase, and in vitro translation using a reticulocyte lysate have all recently been described in detail(14) . The conditions for the post-translational assembly of porin were modified slightly (14) . Mitochondria (200 µg/ml of protein) were incubated for 30 min at 30 °C in a 100-µl mixture. This mixture was comprised of 10 µl of reticulocyte lysate containing in vitro synthesized, radiolabeled yeast porin, an ATP-generating system, and 10 µg/ml valinomycin in an isotonic solution of sorbitol and salts buffered at pH 7.5. In instances when ATP was depleted, the reticulocyte lysate was incubated for 15 min at room temperature with 100 units/ml of apyrase prior to its addition to the mixture, and the ATP-generating system was omitted. The reaction was stopped by dilution with ice-cold 0.6 M sorbitol, 20 mM HEPES (pH 7.5). In most instances, this mixture was treated with 100 µg/ml proteinase K for 15 min on ice, and then the proteinase was inhibited with 2 mM phenylmethylsulfonyl fluoride before the mitochondria were isolated. The conditions for the isolation of mitochondria from the diluted reaction mixture, estimation of membrane binding, and estimation of membrane assembly of yeast porin by its resistance to extraction with 40 mM Na(2)CO(3) (pH 11.5) have already been described(14) . Any deviation from these procedures is indicated in the figure legends. The co-translational assembly reactions using yeast cytosol for synthesis of precursor porin concomitant with its insertion have been described previously(10) .

Construction of Mutant Forms of Yeast Porin

The construction of K19E, K46E, K234E, K234Q, K236E, and K274E mutant porins has been described elsewhere(6) . These sequences were subcloned into pSP65 using the SacI and HindIII sites in the multiple cloning region. K234R (A740G), K234E/K236E (A739G/A745G), K234Q/K236Q (A739C/A745C), and K234R/K236R (A740G/A746G) were constructed using a 945-base pair yeast porin cDNA in which the open reading frame started at base pair 40. Single and double point mutations were introduced using the Altered Sites 1 mutagenesis kit (Promega Corp.) and were screened by DNA sequencing using the Sequenase II kit, version 2.0 (U. S. Biochemical Corp.). These mutants were subcloned into pSP65 using the SacI and BamHI sites in the multiple cloning region, and the mutations were verified again by DNA sequencing. Delta71-116 porin was constructed by introducing HpaI sites into yeast porin at positions 201-208 and 344-351 (counting the first A of the start codon as nucleotide 1) using the Muta-Gene Phagemid in vitro mutagenesis kit (Bio-Rad). The sequences were confirmed using restriction analysis and DNA sequencing using the Sequenase II kit, version 2.0 (U. S. Biochemical Corp.). The deletion was produced by digestion with HpaI and religation. Delta9-156 porin was constructed by digesting the wild-type cDNA (in pSP65) with EcoRV and religation. The structures of these deletion mutants were confirmed by restriction enzyme analysis.

Materials

The 945-base pair wild-type porin cDNA and antibodies against yeast porin were gifts from Dr. G. Schatz (Biozentrum, University of Basel, Basel, Switzerland). Restriction enzymes and other molecular biological reagents were purchased from the Promega Corp. Proteinase K (molecular biology grade), trypsin (grade XIII), apyrase (grade VII), valinomycin, and other biochemical and immunological reagents were obtained from Sigma. Mutagenic and sequencing oligonucleotides were purchased from National Biosciences, Plymouth, MN.


RESULTS

K234E Porin Assembles Poorly into the Mitochondrial Outer Membrane

The assembly of four lysyl to glutamyl mutants of yeast porin (K19E, K46E, K234E, and K274E porins) into the outer membrane of yeast mitochondria was compared with wild-type porin using an in vitro assay. Wild-type and mutant porins were synthesized in a reticulocyte lysate and incubated with mitochondria after ATP depletion or in the presence of an ATP generating system (Fig. 1). The assembly of the fully synthesized precursor porins into the outer membrane was estimated by their protection from 100 µg/ml of proteinase K (Fig. 1, lanes 1 and 3). In our experiments, a degree of insertion was evident for wild-type and each of the mutant porins, even when ATP was depleted (Fig. 1, lane 1). Others have reported that porin assembly into mitochondria is ATP-dependent(3, 11) , and the limited degree of porin insertion seen in Fig. 1, lane 1, was probably due to a failure to sufficiently prevent the synthesis of ATP by the mitochondria. This contention is supported by the observation that porin insertion into isolated outer membranes is completely ATP-dependent(14) . In any case, the assembly of each porin form was markedly improved by the inclusion of ATP in the reaction mixture (Fig. 1, lane 3). In the ATP-supplemented reactions, about 20% of the available wild-type, K19E, K46E, and K274E precursor porins were inserted into the outer membrane (Fig. 1, lane 3). On the other hand, K234E porin was assembled to a much lesser extent whether or not ATP had been depleted. Obviously, mutation of Lys-234 to glutamate affected porin assembly more than the same mutation at Lys-19, Lys-46, or Lys-274. The data in Fig. 1suggested that the lysine at position 234 of yeast porin may have a role in the membrane assembly of this protein; however, other possibilities existed.


Figure 1: The assembly of yeast porin is impaired by mutation of lysine 234 to glutamate. Mitochondria (200 µg/ml) were incubated for 30 min at 30 °C with in vitro synthesized, radiolabeled, wild-type (WT) porin or one of the four indicated porin mutants. The mitochondria were isolated and analyzed by electrophoresis and fluorography. The left lane of the autoradiogram contained an amount of porin or mutant porin equal to 20% of that included in the incubations. Prior to some incubations, ATP was depleted with apyrase at a final concentration of 10 units/ml (lanes 1 and 2). After some incubations, the mitochondria were treated with 100 µg/ml proteinase K (PROT. K) prior to their isolation and analysis (lanes 1 and 3). These conditions are described in more detail under ``Experimental Procedures'' and (14) .



One possibility was that K234E porin was more rapidly degraded during the assembly assay, and if so, this mutant would be less available for membrane insertion. This is unlikely for several reasons. First, when the precursor porins depicted in Fig. 1were incubated in the presence of mitochondria but after ATP depletion, the total amount of each porin (i.e. both mitochondrially associated and free porin) was unchanged (data not shown). Second, the amount of K234E porin available to the mitochondria, as judged by the amount bound to the mitochondria after ATP depletion, was similar to the wild-type (Fig. 1, lane 2). These data indicated that, at least under the in vitro condition of ATP deprivation, K234E porin was not degraded and was as available to the mitochondria as was the wild-type porin. Also, if wild-type, K234E porin or any of the other three porin mutants were incubated in the presence of ATP, but in the absence of mitochondria, none of these proteins suffered detectable degradation (data not shown). These experiments showed that neither wild-type nor any of these mutants were subject to the ATP-dependent mechanisms of proteolysis that are present in the reticulocyte lysate.

It was possible that the K234E porin mutant was a better substrate for proteinase K than were the other types of porin. If this were so, K234E porin might be more readily attacked by this enzyme even if it were properly incorporated into the outer membrane. Experiments were performed to determine whether the data in Fig. 1were due to an artifact of this type. In these experiments, assembly was assessed using protection from 100 µg/ml trypsin (unlikely to be more active against a lysine to glutamate mutation) and resistance to extraction with 40 mM Na(2)CO(3) at pH 11.5 (impervious to changes in primary structure). These data (Fig. 2) show that, under either of these conditions, K234E porin was not as well inserted as was the wild-type. Based on these experiments, it seemed more likely that the mutation of Lys-234 to glutamate was affecting the assembly process. Also in Fig. 2(lane 2 versus lane 4), the assembly of K234E porin was greater as judged by its resistance to Na(2)CO(3) extraction than as judged by its vulnerability to trypsin. It is generally thought that even small tracts of amino acids that are unprotected by membrane lipids may be subject to proteolysis, but substantial portions of a membrane protein can be outside the bilayer without changing its resistance to extraction by Na(2)CO(3). It seemed possible that K234E porin might be partially assembled in the membrane.


Figure 2: Membrane-assembled K234E porin is more sensitive to trypsin and to extraction with Na(2)CO(3) (pH 11.5) than wild-type porin. Mitochondria (200 µg/ml) were incubated for 30 min at 30 °C with in vitro synthesized, radiolabeled, wild-type (WT) porin or K234E porin. The mitochondria were isolated and analyzed by electrophoresis and fluorography. The left lane of the autoradiogram contained an amount of porin or mutant porin equal to 20% of that included in the incubations. Prior to some incubations, ATP was depleted with apyrase at a final concentration of 10 units/ml (lanes 1 and 3). After some incubations, the mitochondria were treated with 100 µg/ml trypsin prior to their isolation and analysis (lanes 1 and 2). After other incubations, the mitochondria were isolated and extracted with 40 mM Na(2)CO(3) (pH 11.5) prior to analysis of the extracted residue (lanes 3 and 4). These conditions are described in more detail under ``Experimental Procedures'' and (14) .



Both Lys-234 and Lys-236 Are Involved in Porin Assembly

Lys-234 is included in an internal pentapeptide, VKAKV (residues 233-237) of yeast porin. It was of interest to see whether the deleterious effects of lysine to glutamate mutation extended to Lys-236. Also, since lysine to glutamate mutations result in a charge reversal, it was important to determine the effects of more conservative substitutions. For these reasons, K236E, K234Q, and K234R mutant porins were compared with wild-type and K234E porin for their ability to assemble into the mitochondrial outer membrane. In these experiments, each porin form was allowed to insert into the membrane in an ATP-supplemented reaction, and the degree of protection of each porin from a range of concentrations of proteinase K (50-200 µg/ml) and its resistance to extraction by Na(2)CO(3) were estimated. As can be seen from the immunoblot in Fig. 3A, lanes 1-4, porin that was endogenous to yeast mitochondria was resistant to proteinase K and to extraction by Na(2)CO(3). In Fig. 3B, in vitro assembled wild-type porin was, like endogenous porin, protected from proteinase K and resistant to Na(2)CO(3) extraction. In contrast, K234E porin was very susceptible to all of the concentrations of proteinase K, while K236E porin was progressively more sensitive to increasing concentrations of the proteinase. K234Q porin was insensitive to the lowest concentration of the proteolytic enzyme but was not as well protected as wild-type at the higher concentrations. As was the case for K234E porin in Fig. 2, the mutants, K234E, K236E, and K234Q, were more resistant to Na(2)CO(3) extraction than to proteinase K digestion. K234R porin was assembled as well as wild-type and was as resistant to proteinase K digestion as it was to Na(2)CO(3) extraction. Clearly, substitution of glutamate for lysine at either position 234 or 236 of yeast porin impaired the membrane assembly of the protein. Mutation of Lys-234 to glutamine also impaired assembly, although not to the extent seen with the K234E and K236E mutations, while mutation to arginine had little or no effect.


Figure 3: Point mutation of either Lys-234 or Lys-236 impairs porin assembly. A, mitochondria (200 µg/ml) were incubated for 30 min at 30 °C in an assembly assay in which in vitro synthesized, radiolabeled, wild-type (WT) porin was replaced by an equivalent amount of reticulocyte lysate mock translation mix. The mitochondria were isolated and analyzed by electrophoresis and immune blotting for endogenous mitochondrial porin. After some incubations, the mitochondria were treated with from 50 to 200 µg/ml proteinase K (PROT. K) prior to their isolation and analysis (lanes 1, 2, and 3). After one of the incubations, the mitochondria were isolated, extracted with 40 mM Na(2)CO(3) (pH 11.5) prior to analysis of the extracted residue (lane 4). The right lane of the immune blot (lane 5) contained mitochondria that were not treated with either proteinase K or extracted with 40 mM Na(2)CO(3) (pH 11.5) prior to analysis. These conditions are described in more detail under ``Experimental Procedures'' and (14) . B, mitochondria (200 µg/ml) were incubated for 30 min at 30 °C with in vitro synthesized, radiolabeled, wild-type porin or the indicated porin mutants. The mitochondria were isolated and analyzed by electrophoresis and fluorography. The left lane of the autoradiogram contained an amount of porin or mutant porin equal to 20% of that included in the incubations. After some incubations, the mitochondria were treated with from 50 to 200 µg/ml proteinase K prior to their isolation and analysis (lanes 1, 2, and 3). After other incubations, the mitochondria were isolated and extracted with 40 mM Na(2)CO(3) (pH 11.5) prior to analysis of the extracted residue (lane 4). These conditions are described in more detail under ``Experimental Procedures'' and (14) .



The observation that K234E, K236E, and K234Q mutant porins were more vulnerable to hydrolysis by proteinase k than to extraction by Na(2)CO(3) suggested that these mutants might be partially assembled into the outer membrane. This contention was supported by evidence presented in Fig. 4. In Fig. 4A, the extent of the assembly of K234E, K236E, and K234Q mutant porins was compared with wild-type porin in an ATP-supplemented assay. As judged by their resistance to 100 µg/ml of proteinase K, none of the mutants assembled as well as wild-type porin. As can be seen in Fig. 4B, after proteinase K treatment of mature wild-type porin, only full-sized porin, one large proteolytic fragment, and smaller fragments that co-migrated with the tracking dye were evident. However, with each of the three mutants (Fig. 4B, lanes 2-4), several additional proteolytic fragments were seen. These data suggested that these mutants were partially membrane assembled but that some regions were exposed and vulnerable to proteolysis.


Figure 4: Membrane-assembled K234E, K236E, and K234Q porins are only partially degraded by proteinase K. A, mitochondria (200 µg/ml) were incubated for 30 min at 30 °C with in vitro synthesized, radiolabeled, wild-type (WT) porin or one of the three indicated porin mutants. The mitochondria were isolated and analyzed by electrophoresis and fluorography. The indicated lanes of the autoradiogram contained an amount of porin or mutant porin equal to 20% of that included in the incubations. After the incubations, the mitochondria were treated with 100 µg/ml proteinase K (PROT. K) prior to their isolation and analysis. These conditions are described in more detail under ``Experimental Procedures'' and (14) . B, the autoradiograhic analyses of the membrane assembly of wild-type porin and the three mutant porins that were described above were over-exposed, and a larger portion of the electrophoretic pattern is depicted. The uppermost band in each lane represents a full-length porin, while the lowermost band co-migrated with the bromphenol blue tracking dye.



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. These mutants were designed to delete either two or eight of the putative membrane spanning tracts of porin (see Fig. 8). As can be seen from Fig. 5, the assembly of the smaller deletion (Delta71-116 porin) was impaired, while the larger deletion (Delta9-156 porin) failed to assemble. It is apparent that porin's N-terminal regions are important for its assembly. However, it is not possible to decide from these experiments whether the impaired assembly of these mutants is due to deletion of informative tracts of amino acids or is a consequence of secondary or tertiary structural changes as a consequence of the deletions.


Figure 8: Putative membrane orientation of yeast porin. In the drawing, membrane-assembled yeast porin is depicted as being comprised of one membrane-spanning amphipathic alpha-helix and 12 membrane-spanning amphipathic beta-strands(6) . The positions at which yeast porin was point mutated are indicated (Lys-19, Lys-46, Lys-234, Lys-236, and Lys-274). The positions at which the deletions occur in Delta71-116 and Delta9-156 porins are also indicated. These deletion mutants are also represented as bars below the drawing. The black regions in these bars indicate the tracts deleted from the drawing above. Delta71-116 and Delta9-156 were designed to remove one beta-hairpin and eight membrane-spanning tracts from membrane assembled yeast porin, respectively.




Figure 5: Deletion of N-terminal tracts of porin impair or prevent its membrane assembly. Mitochondria (200 µg/ml) were incubated for 30 min at 30 °C with in vitro synthesized, radiolabeled, wild-type (WT) porin or one of the two indicated porin mutants. The mitochondria were isolated and analyzed by electrophoresis and fluorography. The right lane of the autoradiogram contained an amount of porin or mutant porin equal to 20% of that included in the incubations. Prior to some incubations, ATP was depleted with apyrase at a final concentration of 10 units/ml (lanes 1 and 2). After some incubations, the mitochondria were treated with 100 µg/ml proteinase K (PROT. K) prior to their isolation and analysis (lanes 1 and 3). These conditions are described in more detail under ``Experimental Procedures'' and (14) .



A Lysine Residue Is Required at Position 234 or 236 for Efficient Membrane Assembly of Porin

It was clear that replacement of either Lys-234 or Lys-236 with glutamate was detrimental to the assembly of porin into the outer membrane and that replacement of Lys-234 with arginine had little or no effect; however, it was not clear why. Were two positively charged amino acids required at these positions? Or would two matched charges be sufficient? Or was at least one lysine residue required? To find out, three double mutants, K234E/K236E porin, K234Q/K236Q porin, and K234R/K236R porin were compared with wild-type in an experiment similar to that shown in Fig. 1. As judged by their binding to the mitochondria, all three double mutants seemed to be as available to the outer membrane as was wild-type porin (Fig. 6, lane 2), and as in Fig. 1, there was some assembly when ATP was depleted (Fig. 6, lane 1). However, even when ATP was present (Fig. 6, lane 3), both K234E-K236E and K234Q-K236Q porins were poorly assembled. These data ruled out the possibility that two negative charges at positions 234 and 236 would mediate membrane insertion as well as positive charges. As might have been expected, K234R/K236R porin was better assembled than either of the other double mutants. On the other hand, K234R/K236R porin was inserted into the membrane to a lesser degree than was wild-type porin. These data indicated that while the positive charges provided by arginine at positions 234 and 236 of porin might, in comparison with glutamate or glutamine, improve membrane assembly, the insertion process was more efficient if lysine was present at one of these positions (i.e. as in K234R porin).


Figure 6: Wild-type porin assembles into the mitochondrial outer membrane better than K234R-K236R porin. Mitochondria (200 µg/ml) were incubated for 30 min at 30 °C with in vitro synthesized, radiolabeled, wild-type (WT) porin or one of the three indicated porin mutants. The mitochondria were isolated and analyzed by electrophoresis and fluorography. The left lane of the autoradiogram contained an amount of porin or mutant porin equal to 20% of that included in the incubations. Prior to some incubations, ATP was depleted with apyrase at a final concentration of 10 units/ml (lanes 1 and 2). After some incubations, the mitochondria were treated with 100 µg/ml proteinase K (PROT. K) prior to their isolation and analysis (lanes 1 and 3). The uppermost band in each lane represents a full-length porin, while the lowermost band co-migrated with the bromphenol blue tracking dye. These conditions are described in more detail under ``Experimental Procedures'' and (14) .



Are Lys-234 and Lys-236 Involved in the Co-translational Membrane Assembly of Porin into the Mitochondrial Outer Membrane?

Since there is good evidence that in yeast mitochondria assembly occurs by co-translational mechanisms(10, 16) , we sought to determine if the effects of the mutations would be maintained in a homologous, co-translational assay. Three mutants (K234E, K236E, and K274E porin) were evaluated in comparison to wild-type porin. In this experiment, yeast cytosol (rather than reticulocyte cytosol) was used to synthesize the precursors, and translation occurred simultaneously with membrane insertion. While wild-type and each of the mutant porins were assembled to some extent (Fig. 7), neither K234E nor K236E porin was as well assembled as K274E or wild-type porin. These data indicated that the influence of the C-terminal mutations at positions 234 and 236 could be felt even during co-translational assembly.


Figure 7: K234E and K236E porins do not assemble as well into the mitochondrial outer membrane in co-translational assays as does wild-type porin. Wild-type (WT) porin and the three indicated mutants were synthesized in a nuclease-treated yeast lysate in the presence of 100 ng/ml chloramphenicol and 200 µg/ml yeast mitochondria. The mitochondria were isolated and analyzed by electrophoresis and fluorography. The left lane of the autoradiogram contained an amount of porin or mutant porin equal to 40% of that included in the incubations. After some incubations, the mitochondria were isolated without further treatment (lane 1). After other incubations, the mitochondria were treated with 100 µg/ml proteinase K (PROT. K) prior to their isolation and analysis (lane 2). These conditions are described in more detail in (10) .




DISCUSSION

Yeast mitochondrial porin has been modeled as a beta-barrel comprised of a single membrane-spanning alpha-helix and 12 membrane-spanning anti-parallel beta-strands (Fig. 8). In this model, the alpha-helix and beta-strands are amphipathic with polar residues projecting into the large aqueous pore that is formed by this protein(6) . Mutation of each of five lysyl residues (Lys-19, Lys-46, Lys-234, Lys-236, and Lys-274) to glutamate was evaluated for its effect on the in vitro assembly of yeast porin into the outer membrane of intact yeast mitochondria. In post-translational assays (i.e. insertion of fully synthesized precursor porins), K19E, K46E, and K274E behaved like wild-type; however, in both post-translational and co-translational (i.e. simultaneous synthesis and insertion of porins) assembly assays, the insertion of K234E and K236E porin was much less than for wild-type porin. Mutation of Lys-234 to glutamine also impaired yeast porin assembly, but to a smaller extent. Replacement of Lys-234 with arginine had little or no effect. Simultaneous mutation of both Lys-234 and Lys-236 to either glutamate or glutamine severely inhibited membrane assembly. Mutation of both of these lysines to arginine impaired porin assembly to a lesser degree. Lys-234 and Lys-236 are part of an internal pentapeptide, VKAKV, that has been proposed to be located toward the cytosolic side of the ninth membrane spanning beta-strand(6) . The mutations in the VKAKV peptide that have been described here can be put into three groups: mutants that insert into yeast mitochondria as well as wild-type (K234R), mutants that do not assemble as well as wild-type (K234Q and K234R/K236R), and mutants that are very poorly assembled (K234E, K236E, K234E/K236E, and K234Q/K236Q). These groupings suggest that in yeast a net positive charge of at least +1 in the VKAKV region is sufficient for assembly; however, two positive charges, one of which is carried by lysine, are optimal. Although the importance of the VKAKV region has only been tested in yeast, this pentapeptide sequence has been conserved in other species (Table 1). In yeast and two other protists, the sequence contains two lysines. In four plant species, two positive charges are carried by an arginine and a lysine. In the four known mammalian sequences, the single positive charge is carried by lysine.



Although positively charged residues in the VKAKV tract appear to be important in the membrane assembly of yeast porin, observations made with three of the point-mutated porins (K234E, K236E, and K234Q) also suggested that other regions of the porin precursor are capable of membrane assembly. Each of these mutants appeared to be more efficiently assembled when resistance to Na(2)CO(3) extraction, instead of protection from proteolysis, was used as a criterion for membrane assembly. Also, after proteolysis of the membrane-inserted forms of these mutants, several porin fragments, which apparently had been protected from the proteinase, remained. Both observations are consistent with the conclusion that these mutants were partially assembled in the membrane. It seems possible that Lys-234 and Lys-236 may be especially important for the membrane insertion of a specific portion of yeast porin (perhaps, a C-terminal tract).

It is clear that the positively charged residues, Lys-234 and Lys-246, participate in the assembly of yeast porin into the mitochondrial outer membrane; however, the way in which they participate is not understood. It has been reported that the assembly of monoamine oxidases, another type of outer membrane protein, depends on a ubiquitin conjugation(17, 18) . However, such a process has not been reported for porin assembly. Furthermore, it is unlikely that ubiquitin conjugation of either Lys-234 or Lys-236 is crucial since mutation of both of these residues to arginine only partially impairs porin assembly. Alternatively, there is good evidence that clusters of positive charges adjacent to hydrophobic alpha-helices ensure correct membrane assembly of some proteins by preventing hydrophobic tracts from traversing the membrane (19, 20, 21) , but again, it is difficult to extend this argument to porin. First, most of the suspected membrane spanning tracts in yeast porin seem to be amphipathic beta-strands instead of hydrophobic alpha-helices. Second, since Lys-234 significantly contributes to the channel properties of yeast porin(6) , this residue, at least, is thought to be in the aqueous channel of the pore rather than at the surface of the bilayer. Also, if these positive charges in porin act only as barriers for the transfer of polypeptide tracts through the bilayer, why is this tract most effective when at least one of these charges is carried by lysine? Another possibility is that ionic interactions of Lys-234 and Lys-236 with other amino acid residues are important for the correct folding of the mature form of porin. While this is not ruled out, it is made less attractive by the fact that Lys-234 has been shown to be in an aqueous environment where such interactions would be expected to be least influential. Finally, the positive residues in the VKAKV region might be recognized and manipulated by proteins that are involved in the membrane insertion of porin. If this were so, it would be easier to understand the preference for lysine over arginine residues in this tract; however, such an interaction has yet to be demonstrated.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants MH 47181 (to R. M.) and GM 35759 (to M. F.) and National Science Foundation Grant MCB-9418257 (to K. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 313-577-6737; Fax: 313-577-6739.


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