(Received for publication, June 15, 1995; and in revised form, September 20, 1995)
From the
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 NaCO
(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.
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 31,000 porin
polypeptide(2) . Although the tertiary structure of eukaryotic
porin is still unknown, it is thought to be largely comprised of
amphipathic
-strands in the form of a
-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
-helix and 12 amphipathic
-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.
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 NaCO
(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) .
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 NaCO
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
CO
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
CO
. 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 NaCO
(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
CO
(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) .
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
NaCO
(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
CO
(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
CO
(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
NaCO
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 (71-116 porin) was impaired, while the
larger deletion (
9-156 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 -helix and 12
membrane-spanning amphipathic
-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
71-116 and
9-156 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.
71-116 and
9-156 were designed to remove one
-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) .
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) .
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) .
Yeast mitochondrial porin has been modeled as a -barrel
comprised of a single membrane-spanning
-helix and 12
membrane-spanning anti-parallel
-strands (Fig. 8). In this
model, the
-helix and
-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
-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
NaCO
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 -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
-strands
instead of hydrophobic
-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.