The Plant Cell, Vol. 10, 1217–1228, July 1998, www.plantcell.org © 1998 American Society of Plant Physiologists
A Cytoplasmic Male Sterility–Associated Mitochondrial
Peptide in Common Bean Is Post-Translationally Regulated
Rodrigo Sarria,a Anna Lyznik,a C. Eduardo Vallejos,b and Sally A. Mackenzie a,1
a Department
b Department
of Agronomy, Lilly Hall, Purdue University, West Lafayette, Indiana 47907
of Horticultural Sciences, University of Florida, Gainesville, Florida 32611
Cytoplasmic male sterility in the common bean plant is associated with a dominant mitochondrial mutation designated
pvs-orf239 (for Phaseolus vulgaris sterility sequence open reading frame 239). The sequence is transcribed in both vegetative and reproductive tissues, but the translation product, ORF239, is present only in reproductive tissues. We
present evidence to support a model of post-translational regulation of ORF239 expression based on the following observations. In organello translation experiments using purified mitochondria from young seedlings demonstrated
accumulation of ORF239 only when a protease inhibitor was included. Proteolytic activity against ORF239 was observed in mitochondrial extracts fractionating with the mitochondrial inner membrane. The DNA sequence encoding a
serine-type protease, similar to the lon protease gene of Escherichia coli, was cloned from the Arabidopsis genome.
The expression product of this sequence demonstrated proteolytic activity against ORF239 in vitro, with features resembling the activity detected in mitochondrial inner membrane preparations. Antibodies generated against the
overexpressed Lon homolog reduced proteolytic activity against ORF239 when added to mitochondrial extracts. Our
data suggest that ORF239 was undetected in vegetative tissue due to rapid turnover by at least one mitochondrial protease that acts against ORF239 post-translationally.
INTRODUCTION
Features common to plant mitochondrial genomes, distinguishing them from other eukaryotes, are most likely the
result of selective constraints imposed by the interactions
of nuclear and cytoplasmic genomes throughout the life cycle of the plant. These features range from genome organization, size variation, and recombinational activity to
nearly all aspects of gene expression (Bonen and Brown,
1993; Wolstenholme and Fauron, 1995). Gene regulation in
plant mitochondria is not yet well understood. Relatively little evidence is available to suggest that transcriptional regulation serves as the predominant means of gene modulation,
although a promoter consensus motif has been described
(Rapp and Stern, 1992; Rapp et al., 1993). Even though
most plant mitochondrial transcripts undergo extensive editing (Hiesel et al., 1994) and, in some cases, cis- and/or
trans-splicing of intron sequences (Wolstenholme and Fauron,
1995), the rate and extent that these processes limit gene
expression are not clear. Likewise, no compelling evidence
has been reported thus far for translational regulation of
plant mitochondrial genes, although considerable information regarding translational activation exists for yeast (Fox,
1996). Here, we present evidence to suggest that one level
of mitochondrial gene regulation that may be important in
plants is post-translational proteolysis.
1 To whom correspondence should be addressed. E-mail smackenz
@purdue.edu; fax 765-494-6508.
Cytoplasmic male sterility (CMS) in higher plants is a maternally inherited phenotype of pollen sterility (Hanson,
1991). Characterization of CMS in several species has
shown that the phenotype arises as a consequence of
dominant mutations in the mitochondrial genome. In each
CMS case examined, the particular mitochondrial mutation
is distinct. The CMS system in common bean differs from
other well-defined systems in several regards. Most important to this study is that the CMS-associated mitochondrial
mutation in common bean, designated pvs-or f 239 (for
Phaseolus vulgaris sterility sequence open reading frame
239), is transcribed in both vegetative (young seedling) and
reproductive tissues (Chase, 1994). The transcript of pvsor f 239 has been demonstrated to associate with polysomes at both stages (Chase, 1994); however, its product,
ORF239, accumulates only in reproductive tissues (Abad et
al., 1995). The ORF239 product, which is z27 kD in size,
ultimately is localized within the callose layer of the premeiotic pollen mother cell wall (Abad et al., 1995; He et al.,
1996).
The observation of tissue-specific expression of a dominant mitochondrial mutation led us to investigate further the
means of ORF239 regulation. Here, we present evidence to
suggest that ORF239 expression in CMS common bean is
regulated post-translationally, and we identify one mitochondrial protease in plants that appears to be involved in
ORF239 turnover.
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RESULTS
orf239 Encodes a Translationally Competent Transcript
in Vegetative (Young Seedling) Tissues in CMS-Sprite
Mitochondria of the cytoplasmic male-sterile line of common
bean, CMS-Sprite, perform transcription of the sterility-associated mitochondrial sequence pvs-orf239 in both vegetative
and reproductive tissues (Chase, 1994). The DNA sequence
of pvs-orf239 encodes a polypeptide of z27 kD (Johns et
al., 1992). Yet, the translation product of this mutation,
ORF239, is detected only during the development of reproductive organs (Abad et al., 1995). To test whether the
pvs-orf239 transcript detected in vegetative tissues is translationally competent, in organello translations in the presence of 35S-cysteine and an energy source were performed
using purified mitochondria from etiolated 10-day-old seedlings. Figure 1 shows that mitochondria from CMS-Sprite
seedlings produced no detectable ORF239 protein upon immunoprecipitation by using polyclonal anti-ORF239 anti-bodies.
However, when mitochondrial incubations of CMS-Sprite
included a protease inhibitor, an z30-kD mitochondrial
polypeptide was immunoprecipitated that comigrated with
the in vitro–translated ORF239 control (Figure 1B). The
specificity of the anti-ORF239 antibodies had been extensively tested previously (Abad et al., 1995; He et al., 1996).
Of the inhibitors used, all known to be membrane permeable, vanadate acted as the most effective inhibitor of
ORF239 turnover, whereas o-phenanthroline and N-ethylmaleimide (NEM) were less effective (Figure 1B). The inhibitor
hemin interfered with in organello translation (data not
shown) so that it was not possible to determine its effect on
ORF239 stability.
An unexpected observation emerged from these assays;
although the ORF239 monomeric form immunoprecipitated
in small amounts upon in organello translation, two larger
forms of the protein were evident in greater amounts. We
identified these higher molecular mass bands as ORF239.
These bands most likely represent multimeric forms based
on the following observations. The larger forms immunoprecipitated with anti-ORF239 antibodies in both in organello
translations and in vitro translations (Figure 1C). Protein gel
blot analysis of in organello and in vitro translation products
indicated strong cross-reactivity of the larger forms with
anti-ORF239 antibodies (data not shown). The accumulation
of the higher molecular mass forms demonstrated a direct
correlation with accumulation of the ORF239 monomeric
form, and proteolytic turnover of ORF239 was proportional
to turnover of the larger forms (Figure 1C and data not
shown). A strong propensity to aggregate is routinely observed in the purification of ORF239 (data not shown).
The results of the in organello translation experiments
demonstrate that the pvs-orf239 transcripts detected in
mitochondria from vegetative tissues are competent for
translation and suggest that some degree of regulation of
expression in vivo might be post-translational. Furthermore,
the observation of several labeled proteins accumulating in
mitochondrial incubations without the addition of protease
inhibitors (Figure 1A) suggests that rapid proteolysis under
these conditions has some degree of specificity to the
ORF239 protein.
Plant Mitochondrial Extracts Contain Proteolytic Activity
against the ORF239 Substrate
To characterize further the proteolysis of ORF239 in CMSSprite, we identified the location of the protease activity in
the organelle and determined some of the properties of the
protease. For this purpose, purified mitochondria from
CMS-Sprite etiolated seedlings were fractionated to matrix,
inner membrane, and outer membrane components. The
proteolytic activity of these fractions was tested using as
substrates in vitro–transcribed/translated 35S-ORF239 and
resorufin-labeled casein, a well-defined protease substrate.
Proteolytic activity against 35S-ORF239 was quantitated by
the amount of acid-precipitable counts per minute accumulated after translation of the in vitro–transcribed/translated
ORF239 (Table 1) and by SDS-PAGE (data not shown). To
determine the proteolytic activity against resorufin-labeled
casein, the amount of resorufin remaining was quantitated
spectrophotometrically after trichloroacetic acid (TCA) precipitation. Figure 2 and Table 1 show that the proteolytic activity was associated primarily with the inner mitochondrial
membrane. Fractionations involving sonication or more extensive detergent treatment (.15-min incubation with lubrol)
of the inner membrane consistently resulted in the detection
of activity in the matrix fraction (data not shown). These results suggest that the association of the detected protease
activity with inner mitochondrial membrane is easily disrupted and likely does not involve membrane integration.
The protease activity detected in inner membrane fractions
tested against a casein substrate demonstrated ATP dependence and sensitivity to NEM, phenylmethylsulfonyl fluoride
(PMSF), vanadate, and EDTA (Figure 2); these properties can
be indicative of a serine-type protease. Furthermore, although some sensitivity of the protease to o-phenanthroline
was detected, this sensitivity was not influenced by subsequent addition of Mn 21 (data not shown), suggesting that
detected activity was not likely due to the presence of a
metalloprotease. Both serine-type and metalloproteases
have been identified in mitochondria of animal and yeast
systems (Rep and Grivell, 1996), and we cannot exclude the
possibility that metalloprotease activity may also be present
in inner membrane fractions.
Variable results and sometimes undetectable levels of
turnover were obtained when mitochondrial fractions were
incubated with 35S-labeled in vitro–transcribed/translated
ORF239 post-translationally (Table 1, IVT-PT ORF239), requiring a more sensitive assay of protease action. When
the in vitro–transcribed/translated ORF239 substrate was
Mitochondrial Proteolysis in CMS Bean
1219
heated to 658C before incubation, substrate turnover rate increased. This change in turnover rate suggested either that
the ORF239 protein precipitates rapidly and is unavailable to
the protease or that the rate of proteolysis is dependent on
structure of the substrate, as has been observed by others
(Wagner et al., 1994; Van Melderen et al., 1996). The first
possibility was disregarded because ORF239 is completely
undetectable in vivo, even when sensitive ELISA procedures
are used (Abad et al., 1995). We investigated the second
possibility and tested whether the proteolytic activity detected in purified mitochondria would act cotranslationally.
The addition of prepared inner-membrane mitochondrial extracts to in vitro–transcribed/translated ORF239 concurrent
with its translation (see Methods) resulted in the increase
of ORF239 turnover (Table 1, Inner membrane fraction).
This result is consistent with our current observations of
highly rapid ORF239 turnover in organello. Although such in
vitro observations cannot be assumed a priori to reflect in
vivo events without further investigation, our results suggest
that the efficient turnover of the ORF239 substrate in vivo
may be the consequence of cotranslational proteolysis or
particular chaperonin activity that could not be duplicated in
our in vitro assay. Thus, it is important to note that the results of proteolysis assays presented in Table 1 involving
cotranslational turnover of ORF239 present results in terms
of accumulation of ORF239 translation product rather than
its degradation. Such an assay required the inclusion of additional controls that are detailed in Methods.
A Mitochondrial Protease Is Encoded in the Plant
Nuclear Genome
Figure 1. In Organello Translation Products of Mitochondria Prepared from CMS-Sprite and Isonuclear Fertile Revertant Lines.
The ATP-dependent protease activity associated with the
inner mitochondrial membrane in CMS-Sprite has properties
in common with the Escherichia coli–like Lon protease detected in human and yeast mitochondria (Gottesman, 1989;
Goldberg, 1992; Wang et al., 1993, 1994; Suzuki et al., 1994;
Van Dyck et al., 1994). Based on this observation, we tested
whether the plant protease might be a lon homolog. Using
degenerate primers designed from the most conserved
Mitochondria were prepared from etiolated young seedling tissues,
and in organello reactions were incubated in the absence and presence (1) of the protease inhibitors vanadate (Van; 4 mM), o-phenanthroline (o-Phen; 5 mM), and N-ethylmaleimide (NEM; 2 mM).
(A) Total in organello translation products from fertile revertant
WPR-3 (isogenic to CMS-Sprite but lacking pvs-orf239) produced in
the absence and presence of an added protease inhibitor (vanadate,
1Van). No Inhib indicates no inhibitor.
(B) Translation products from CMS-Sprite immunoprecipitated with
anti-ORF239 antibodies and a control lane (from the same gel; No Inhib
[no inhibitor]) containing in vitro–transcribed/translated ORF 239
(IVT-ORF239) immunoprecipitated with the anti-ORF239 antibody.
The expected location of ORF239 is indicated at right, although two
additional higher molecular mass proteins also precipitated. These
larger proteins, which are present in both in vitro–transcribed/translated and in organello translation products, appeared to be newly
synthesized, to cross-react with anti-ORF239 antibodies, and to accumulate only in the presence of protease inhibitors. We believe
these larger products represent multimeric or aggregated forms of
the ORF239 product, and they demonstrate a similar susceptibility
to proteolysis.
(C) The first two lanes contain products recovered with the preimmune (1Preim) and anti-ORF239 antibodies after in vitro transcription and translation of the ORF239 clone. The third lane shows the
recovery of ORF239 after immunoprecipitation of in vitro–translated
mitochondrial RNA by using anti-ORF239 antibodies.
In (A) to (C), numbers at left indicate molecular mass standards in kilodaltons.
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Table 1. Percentage of Accumulation of in Vitro–Transcribed/Translated ORF239 a Incubated in the Presence of Mitochondrial Fractions and in
Vitro–Transcribed/Translated AtLON
Protease Source
No Inhibitors
1 Vanadate b
1 o-Phenanthroline b
1 Anti-AtLON b
Preincubation
Anti-AtLON c
IVT-PT ORF239 d
Inner membrane fraction
Matrix fraction
IVT AtLON
No fraction added
47 6 5.1
110 6 4
64 6 7.2
100 6 0.9
84 6 3.2
105 6 3.3
77 6 4
—g
85 6 5.5 (72 6 7) e
108 6 1.2
67 6 5
—
81 6 4.4
ND f
87 6 2.8
—
65 6 3.5
ND
73 6 4.3
—
98 6 8
99 6 5.3
98 6 4.1
—
a Refers
to proteolysis of in vitro–transcribed/translated ORF239 while the translation reaction was performed. Values are presented as the percentage of counts that are accumulated after 90 min. Average and standard error values based on two experiments are shown. Accumulation of
35S-ORF239 was determined by TCA-precipitable counts. The accumulation of in vitro–transcribed/translated ORF239 in the absence of any
added mitochondrial fraction was considered to be the 100% accumulation value of in vitro–transcribed/translated ORF239 and was used as the
basis of comparison for the other treatments.
b (1), added to incubation mixture.
c Anti-AtLON antibodies were preincubated with in vitro–transcribed/translated AtLON before the reaction.
d IVT-PT ORF239 refers to the substrate added post-translationally. In this case, counts must be interpreted as counts remaining after proteolysis of a fuller translated in vitro–transcribed/translated ORF239 product rather than counts accumulated during translation.
e In the course of our experiments, we observed that o-phenanthroline contributed to translational efficiency of the in vitro–transcribed/translated
reaction, even when the protease was absent from the reaction. Consequently, the value in parentheses represents the adjusted value subtracting the contribution of o-phenanthroline to the translation reaction.
f ND, not determined.
g (—) indicates that the experiment was not conducted.
regions of the eukaryotic lon homologs, we were able to use
polymerase chain reaction (PCR) to amplify a discrete product from the genome of Arabidopsis. The rationale for the
amplification of the Arabidopsis sequence, rather than the
common bean sequence, was based on the amenability of
Arabidopsis to subsequent transformation and mutagenesis
experiments. Furthermore, antibodies raised against the Arabidopsis gene product are likely to cross-react with the
common bean product because Lon homologs are highly
conserved.
The 1.8-kb degenerate PCR amplification product from
Arabidopsis (data not shown) was cloned and used to probe
an Arabidopsis cDNA library. A selected cDNA clone of 3.1
kb was identified and sequenced to confirm homology to
the LON genes previously identified in other species. A rapid
amplification of cDNA ends (RACE) and PCR procedure was
used to obtain the 59 end of the gene. Multiple primers were
used in the RACE-PCR amplifications to ensure that the
complete 59 sequence had been obtained (data not shown).
The lon-homologous sequence from Arabidopsis has GenBank accession number ATU88087.
Sequence analysis identified an ORF of 2825 bp encoding
a polypeptide of 110 kD. Figure 3 shows that the polypeptide has a significant amino acid sequence similarity to the
E. coli Lon protease (61.1%) and its homolog in yeast
(64.3%) and to a recently identified sequence in maize
(75%). The first ATG after the start of transcription presents
a conserved Kozak-like sequence (Kozak, 1986) at this site
and also marks the beginning of a putative mitochondrial
transit sequence of 55 amino acids. A well-conserved ATP
binding domain (positions 464 to 471) and a catalytic do-
main (positions 839 to 847) with the requisite serine residue
(position 842) also demonstrate high sequence conservation
(Figure 3). No obvious membrane-spanning domains were
evident in the sequence. Computer analysis with the PSORT
program (Nakai and Kanehisa, 1992) predicted most probable localization of the encoded gene product to the mitochondrial matrix with a discrimination value of 0.614; a
discrimination value of 0.317 was obtained for localization to
the mitochondrial inner membrane, intermembrane space,
and outer membrane. These results also support our hypothesis that a mitochondrial lon homolog is encoded within
the plant nuclear genome.
The selected cDNA clone representing a putative lon homolog from Arabidopsis ( AtLON ) was used to probe the
common bean genome. DNA gel blot analysis with moderately stringent conditions (described in Methods) allowed us
to detect hybridization to what appeared to be a single-copy
locus in the common bean genome with several restriction
enzymes (data not shown). To confirm that the hybridizing
band(s) in common bean represented a single locus, we
mapped the locus based on two distinct restriction fragment
length polymorphisms (RFLPs) detected between parental
bean lines Calima (C) and Jamapa (J) with the enzymes
HaeIII (fragments of 6.6 kb [C] and 8.0 kb [J]) and HpaII
(fragments of 10.7 kb [C] and 9.1 kb [J]). Both RFLPs
mapped to linkage group A, z1.4 centimorgans (LOD 17.7)
away from marker Bng23 on the common bean map developed by Vallejos et al. (1992). Mapping was conducted using a recombinant inbred population of 76 lines. These data
confirm that a common bean lon-homologous gene is
present as a single-copy locus. Moreover, Figure 4 shows
Mitochondrial Proteolysis in CMS Bean
the results of RNA gel blot hybridization experiments using
the homologous cDNA clone as a probe. The transcripts detected were of the expected lengths of 3.2 kb in Arabidopsis
and 3.1 kb in common bean leaf tissues. This observation
further confirmed that we had identified the full-length cDNA
sequence from Arabidopsis.
Evidence That Some Portion of Mitochondrial
Proteolytic Activity against ORF239 Is
Encoded by the AtLON Gene
In vitro transcription and translation reactions were conducted with the cloned AtLON sequence to test for proteolytic activity. The in vitro–transcribed/translated AtLON
product was incubated with both resorufin-labeled casein
and in vitro–transcribed/translated 35S-ORF239 substrates
to characterize activity relative to that observed using bean
mitochondrial extracts. Figure 5 and Table 1 (see IVT AtLON)
demonstrate that similarities were observed between the IVT
Figure 2. Protease Activity Detected in CMS-Sprite Mitochondrial
Inner Membrane Fractions.
Resorufin-labeled casein (90 mg per reaction) was incubated as substrate with purified and fractionated mitochondrial extracts (10 mg
per reaction) at 378C for 120 min in the presence of various protease
inhibitors or under conditions of ATP depletion. Substrate turnover
was assayed by measuring absorbance of TCA-nonprecipitable
peptide. The data used are the average of two experiments, and the
bars indicate standard errors obtained at each time point.
1221
AtLON product and mitochondrial fractions in ATP dependence, response to different inhibitors, casein degradation,
and cotranslational turnover of the ORF239 substrate.
It was noted that activity of the LON in vitro–transcribed/
translated product was much lower than was the activity detected in mitochondrial extracts. In vitro proteolysis assays
using in vitro–transcribed/translated protease product have
not been reported previously using a eukaryotic Lon homolog, and in vitro assays have been problematic using the
E. coli lon gene (Zehnbauer and Markovitz, 1980; Goff and
Goldberg, 1987; Sonezaki et al., 1994); thus, we have limited
basis for comparison with other systems. It is reasonable to
assume that important mitochondrial components are absent from our in vitro reaction. Furthermore, it was not possible to quantify accurately the amounts of protease present
in mitochondrial extracts relative to that produced in vitro.
These factors most likely contribute to the lowered activity
observed. Nevertheless, our results confirm that the gene
cloned encodes a protease, and its similarity in behavior to
mitochondrial preparations supports our hypothesis that the
cloned gene contributes at least a portion of the protease
activity detected in mitochondrial extracts.
Rabbit polyclonal antibodies were raised against an E. coli
overexpression product derived from a partial clone of the
AtLON sequence (representing bases 304 to 1330 of AtLON
sequence in the database). Protein gel blot analysis confirmed cross-reactivity of the prepared antibodies (antiAtLON) against the cloned overexpression product used as
antigen (data not shown) and against an z100-kD polypeptide in common bean mitochondrial protein preparations, as
seen in Figure 6. Crude cell fractionations prepared from
bean tissues by using differential centrifugation to nuclear,
plastid, and mitochondrial fractions resulted in detection of
.90% of the Lon homolog within the mitochondrial fraction
(data not shown). Fractionation of highly purified mitochondrial preparations from common bean to matrix, inner membrane, and outer membrane components demonstrated that
the cross-reacting protein was detected mainly in inner
membrane fractions. Coincubation of mitochondrial inner
membrane fractions with anti-AtLON antibodies during the
assays for protease activity against in vitro–transcribed/
translated ORF239 substrate resulted in an increase in accumulation of the ORF239 translation product, rising from
47 to 81% (Table 1). Incubation of the in vitro–transcribed/
translated AtLON product with antibodies during incubation
with in vitro–transcribed/translated ORF239 resulted in a
similar increase in substrate accumulation, rising from 64 to
87% (Table 1). These results imply that antibodies against
the homolog AtLON interfere with the protease activity detected in common bean mitochondria.
To test this interaction more directly, competition experiments were conducted by preincubating anti-AtLON antibodies with the in vitro–transcribed/translated AtLON antigen
before their addition to the protease reaction. The addition of
the preincubated anti-AtLON antibodies to the protease reaction had measurably less influence on proteolysis (Table 1),
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The Plant Cell
suggesting that the antibody interacts specifically with the
mitochondrial protease. However, preincubation of the antibodies with the in vitro–transcribed/translated AtLON antigen
did not eliminate completely the effect of the antibodies; this
is likely due to the fact that antibodies were raised against a
truncated form of the antigen, minus the active site, so that
antibody-to-antigen cross-reactivity was less efficient. This
assumption is based on the similar results obtained with both
mitochondrial extracts and in–vitro transcribed/translated
AtLON proteolysis assays. These experiments provide further
evidence that the AtLON homolog that was cloned is responsible, at least in part, for the proteolytic activity detected in
mitochondrial extracts.
DISCUSSION
Tissue-specific expression of plant mitochondrial genes has
not been reported widely, although some recent evidence
does suggest possible transcriptional modulation in different
tissues (Li et al., 1996). To our knowledge, the common bean
pvs-orf239 sequence is the only demonstrated example of a
sequence regulated post-translationally or cotranslationally
in plant mitochondria. Notwithstanding, protein turnover likely
represents a significant mode of mitochondrial gene regulation in higher plants, based on the following observations.
Over the past several years, it has become increasingly evident that the majority of plant mitochondrial genes produce
transcripts that require editing before proper translation (Hiesel
et al., 1994). Only recently, however, has it become clear that at
least some of these transcripts are translated before editing (Lu
et al., 1996). Because these aberrant translation products do
not appear to accumulate in the mitochondrion, it is likely that
they are targeted for rapid degradation. Likewise, the plant
mitochondrial genome is unique in its propensity to undergo
intergenic and intragenic recombination as well as DNA insertions and rearrangements (Wolstenholme and Fauron, 1995).
Several examples exist whereby such genomic rearrangements generate unusual ORFs that are translated (Dewey et
al., 1987; Nivison and Hanson, 1989; Krishnasamy and
Makaroff, 1994; Abad et al., 1995). The pvs-orf239 appears
to be such a sequence, and proteolysis is an important
means of regulating its expression.
Figure 3. Amino Acid Sequence Comparison of the Arabidopsis
Lon Homolog (AtLON) with Products of Other Previously Cloned lon
Genes.
Sequence of a full-length cDNA clone (3331 bp; GenBank accession
number ATU88087) reveals an ORF that encodes 941 amino acid
residues. The deduced amino acid sequence was aligned with Lon
homologs from maize (Zmays2), yeast, and E. coli. The AtLON peptide contains highly conserved ATP binding and catalytic domains
(underlined). The serine residue (asterisk), which is typical of this
serine protease, is also present in the catalytic domain. Black boxes
indicate amino acid identity in relation with AtLON. Spaces (no
amino acid) optimize alignment.The maize sequence, with GenBank
accession number U85495, has been reported by Barakat et al. (1998).
Mitochondrial Proteolysis in CMS Bean
1223
ORF239 would be unique to the plant mitochondrial LON
protease. Because ribosomes localize to the inner mitochondrial membrane (Spithill et al., 1978; Marzuki and
Hibbs, 1986; McMullin and Fox, 1993), the observed membrane association most likely would facilitate its activity in
association with active ribosomes.
An alternative interpretation of our results, however, would
implicate a necessary chaperonin function missing from
our assay. It is clear that LON-mediated proteolysis is dependent on secondary structure of the substrate (reviewed
in Gottesman et al., 1997). The possibility exists that our in
vitro reaction conditions do not supply adequately the necessary chaperonin activity. Thus, the observed cotranslational
Figure 4. Expression of a lon Homolog in Common Bean and Arabidopsis.
RNA gel blot analysis of total mRNA prepared from Arabidopsis (5
mg) and CMS-Sprite (10 mg) leaf tissues demonstrates hybridization
with 3.2- and 3.1-kb transcripts, respectively, when probed with the
cloned AtLON sequence. Numbers at left indicate molecular lengths
in kilobases.
Another example of post-translational regulation in mitochondria is its influence on the coordination of nuclear-mitochondrial gene expression. It is well known that mitochondrially
encoded subunits of the electron transport apparatus, when
expressed in the absence of their nuclear-encoded counterparts, demonstrate unusually rapid turnover (reviewed in Rep
and Grivell, 1996). The question we have not yet addressed is
whether the protease that we have characterized here is involved in all of these necessary protein turnover functions.
The Lon protease in E. coli is an ATP-dependent, serinetype protease that is responsible for much of the protein
turnover that occurs due to mistranslation or misassembly
of proteins (Chung and Goldberg, 1981). Homologous genes
in eukaryotes, bearing significant similarity to the E. coli lon
sequence, have been cloned from the human and yeast genomes and demonstrated to encode a mitochondrial protease. The function of this protease in eukaryotes and its
natural substrates have not yet been fully determined. In
yeast, the mitochondrial Lon homolog has been shown to be
essential to respiratory function, and its loss results in a petite phenotype (Suzuki et al., 1994; Van Dyck et al., 1994). In
the cases of human and yeast, LON protease activity is
present in matrix fractions (Suzuki et al., 1994; Van Dyck et
al., 1994; Wang et al., 1994). Consequently, the association
of the plant Lon homolog with the inner mitochondrial membrane was unexpected. Upon further analysis, however, this
observation is, perhaps, fully in keeping with its apparent
capability to target substrates for cotranslational degradation. Our data in support of cotranslational activity are based
strictly on in vitro analyses, and it will be technically challenging to determine definitively whether the plant LON protease acts cotranslationally in vivo. If our observations do
mimic in vivo activity, the cotranslational turnover of
Figure 5. Proteolytic Activity Is Detected in in Vitro–Transcription/
Translation Products from the Cloned Plant LON Homolog.
Incubation of resorufin-labeled casein substrate (90 mg per reaction)
with in vitro–transcribed/translated LON prepared using the fulllength AtLON clone (10 mL of product per reaction) demonstrated
proteolytic activity. Substrate turnover was monitored spectrophotometrically as absorbance of TCA-nonprecipitable peptide. The addition of protease inhibitors resulted in a pattern of response similar
to that observed using mitochondrial extracts (see Figure 2). Note
that proteolytic activity observed by using in vitro–transcribed/translated LON in the labeled-casein assay was significantly lower than
was the activity detected using mitochondrial extracts; this is most
likely accounted for, in part, by difficulties in accurately quantifying
the protease present in the in vitro–transcribed/translated AtLON
preparations versus mitochondrial extracts. The values used correspond to the average of two experiments, and the bars in each time
point correspond to the standard error.
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Figure 6. Protein Gel Blot Analysis of Fractionated Common Bean
Mitochondrial Proteins.
Mitochondria from etiolated CMS-Sprite seedlings were purified
over Percoll gradients and fractionated by gentle detergent treatment, and the proteins were separated by SDS-PAGE. Protein gel
blot analysis was performed with anti-AtLON polyclonal antibodies
prepared against the overexpression product of the cloned AtLON
homolog. A protein of z100 kD in the inner membrane (IM) fraction
was detected. Note that a small amount of the protein is detectable
in matrix (MAT) and outer membrane/inner membrane space (OM)
fractions as well. This is assumed to be the consequence of slight
cross-contamination of the fractions during preparation.
activity in vitro would simply signal the necessity of proper
folding to the reaction.
We have identified only one component of the plant mitochondrial proteolysis system, and we do not assume this to be
the only protease present. However, others investigating plant
mitochondrial proteases that are involved in protein turnover
have detected little or no activity in mitochondrial matrix fractions; nearly all activity reported to date has been localized to
the inner membrane fractions (Knorpp et al., 1995). Furthermore, it is clear that activity in plant mitochondrial preparations is difficult to detect using in vitro assays. It is for this
reason that we have presented data from proteolysis assays
by using both resorufin-labeled casein, a highly sensitive
assay for substrate turnover, and in vitro–transcribed/translated ORF239. The low activity detected, relative to that reported in human or yeast systems tested, may be the
consequence of less than optimal reaction conditions coupled with the unique features of the plant mitochondrial LON
protease.
The results of this study demonstrate that plant mitochondria contain a LON-like protease that is present in mitochondria from vegetative tissues and is able to degrade the
mitochondrial sterility-associated mutant protein ORF239. If
our observations account, at least in part, for the absence of
ORF239 in seedling tissues, what remains to be deduced
are the events occurring during microsporogenesis. During
pollen mother cell development, the ORF239 protein is apparently stable, accumulating within the callose layer and
the primary cell wall. We have not yet determined whether
the LON protease is expressed or active during pollen
mother cell development. Although protein gel blot analysis
of proteins prepared from young, premeiotic and postmeiotic buds indicates that LON is present (data not shown), it
is not feasible to achieve sufficient resolution by using immunocytological techniques to evaluate expression of the
protease in the pollen mother cell itself. We currently are implementing transgenic analyses to examine in more detail
the pattern of lon promoter activity during floral development. However, our observation of tissue-specific differences in AtLON transcript levels does support the notion of
developmental influence on expression.
Other nuclear suppressors of pvs-orf239 expression,
known as fertility restorer genes, have been identified in
common bean. One, designated Fr2 (Mackenzie, 1991),
most likely acts post-transcriptionally, as does the AtLON
homolog; transcription of the pvs-orf239 region appears to
be unaffected in an Fr2-restored line (Chase, 1994), but the
ORF239 protein is no longer present in developing anthers
(Abad et al., 1995). Therefore, one question raised during
the course of this study was whether the lon homolog and
Fr2 might, in fact, be one and the same. Genetic mapping of
the lon homolog indicated that the locus present on linkage
group A of the bean genome is not allelic to Fr2, which is located on linkage group K (Jia et al., 1997). However, we have
not excluded the possibility that Fr2 also might regulate pvsorf239 expression post-translationally or perhaps interact
with the Lon homolog to activate proteolysis specifically
during anther development. These investigations continue.
METHODS
Mitochondrial Preparations and Fractionations
Mitochondria were isolated from 10-day-old, dark-grown cytoplasmic male sterility (CMS)–Sprite (Phaseolus vulgaris) seedlings, as reported by Forde et al. (1978), using a 26% Percoll gradient for
purification. Mitochondria were resuspended in one-tenth of the final
pellet volume (approximate protein concentration of 40 mg/mL). Subfractionation was conducted using detergent treatment, as described by Ragan et al. (1987). More extensive detergent treatment
of mitoplast was performed by extending lubrol incubation time to 15
min or by increasing lubrol concentration to 0.2 mg/mL.
DNA Cloning, Library Screenings, and Polymerase
Chain Reactions
Degenerate primers 59-ATHT TRGAYGARGAYCAYTAYGG-39 and 59GCTYGGYCCGTCCT TYGGRGT-39 were used to amplify a conserved region of the lon protease gene. (H, R, and Y designate variable nucleotide sites.) The 1.8-kb fragment obtained was cloned in
the PCRII vector (Invitrogen, Carlsbad, CA). BLAST analysis was performed to determine identity of the fragment. This fragment was used
subsequently to screen a lPRLZ, Ziplox derivative (Bethesda Research
Laboratories, Gaithersburg, MD; see Focus, volume 14, pages 76 to
79) Arabidopsis thaliana (ecotype Columbia, nonglaborous) cDNA library that was provided by the Arabidopsis Research Center (ABRC,
Columbus, Ohio). The library contains SalI-NotI cDNA inserts and
was generated by mixing equal amounts of mRNA from several plant
tissues and stages.
The 3.1-kb Ara12 clone was isolated and excised as described by
D’Alessio et al. (Bethesda Research Laboratories; Focus, volume 14,
Mitochondrial Proteolysis in CMS Bean
pages 76 to 79) and subcloned for double-strand DNA sequence
analysis. The insert from Ara12 was cloned into the pBS1 vector
(Stratagene, La Jolla, CA) as an SalI-XbaI fragment (Aralon12). The
missing 59 end portion of the gene was obtained by using the 59 and
39 rapid amplification of cDNA ends (RACE) kit from Boehringer
Mannheim. Primers 59-TGAAATCTGAGCAAGTGTGCCAAC-39 (SP1),
59-TCAGCTGATGGGTCATCCT TCAGA-39 (SP2), and 59-CTATGTGGCACCGGCAATGCTAGA-39 (SP3), located 365, 276, and 136 bp,
respectively, from the 59 end of Aralon12, were used. A 450-bp fragment was obtained, cloned in the PCRII vector (clone 210F), and sequenced.
To determine the accuracy of the sequence obtained at the new 59
end, a second RACE reaction was performed using SP3, and two additional upstream primers designed from the 210F sequence were
obtained. They are 59-TCTCCAT TGGTAGGCTCAGA-39 (SP4) and
59-AGGCAAGAAACCAT TGGAG-39 (SP5). Primers SP4 and SP5
were located 300 and 50 bp from the 59 end of clone 210F, respectively; SP4 spans the 39 end of clone 210F and the 59 end of clone
Ara12. The ATG start codon for the prepeptide was bordered by a
highly conserved Kozak sequence (Kozak, 1986). Prediction of a putative mitochondrial targeting sequence was made using the PSORT
program (World Wide Web) by employing the discriminant analysis
from values of partial amino acid composition (Nakai and Kanehisa,
1992).
The clone designated lon, in which most of the putative transit sequence was not included, was made by amplifying the sequence
between the primers 59-GGGCATGCCT TCTCCAGTGGAGTCTCCATT-39 and SP3 from clone 210F. An SphI site was introduced at the
59 end of the upstream primer. The polymerase chain reaction (PCR)
product was then SphI-BlnI digested and cloned into the Aralon12
construct. Primer 59-GAATTCGAGCCTACCAATGGAGAGGCGGCG-39
(ExplonF) containing an EcoRI site and primer 5 9-GTCGACTCACGT TAAACTCGCT TGA-39 (ExplonR) containing an SalI site were
used to amplify from 304 to 1330 bp of the gene sequence. The PCR
product was EcoRI-SalI digested and cloned into the pMALp2 expression vector (New England Biolabs, Beverly, MA). The clone generated was named Explon. Pvs-orf239 was cloned as a KpnI-SacI
fragment into pBluescript SK2 (Stratagene).
DNA sequencing was done using the ALFexpress DNA sequencer
system from Pharmacia Biotechnology. Sequence analysis was conducted using Expassy tools (World Wide Web). All PCR reactions
were performed in an Amplitron I thermocycler (Thermoline, Dubuque, IA). Pwo DNA polymerase used for the PCR reactions was
purchased from Boehringer Mannheim.
RNA Gel Blot Analysis
Plant total RNA extractions were performed as described by
Pawlowski et al. (1994). Samples (5 to 10 mg per lane) were separated in a formaldehyde-containing 1% agarose gel, as described by
Sambrook et al. (1989). Hybridization buffer was 0.25 M NaHPO4, pH
7.2, 7% SDS, and 1 mM EDTA. Hybridization conditions were 658C
for 16 hr, followed by two washes for 20 min each with 20 mM
NaHPO4, pH 7.2, 1% SDS, and 1 mM EDTA at 658C.
1225
sisted of 76 lines. Restriction fragment length polymorphism (RFLP)
analysis (DNA extraction, DNA gel blotting, probe labeling, hybridizations, and autoradiography) was conducted essentially as described
earlier (Vallejos et al., 1992). Hybridizations with AtLON were conducted at 508C for 16 hr; after hybridization, the blots were washed
successively for 15 min at 508C in 2 3, 1 3, and 0.1 3 SSPE (1 3
SSPE is 0.15 M NaCl, 10 mM sodium phosphate, and 1 mM EDTA,
pH 7.4) containing 0.1% SDS. MAPMAKER 3.0 (Lander et al., 1987;
Lincoln et al., 1992) was used to analyze the linkage relationships of
the AtLON homolog with 36 previously mapped DNA marker loci distributed throughout the common bean genome (Vallejos et al., 1992;
Boutin et al., 1995).
In Vitro Transcription and Translation
Clones AtLon and orf239 were translated using the coupled transcription/translation (TNT) reticulocyte lysate system (Promega,
Madison, WI). DNA template (1 mg) orf239 was transcribed using T7
RNA polymerase, and AtLon was transcribed using T3 RNA polymerase. Incubation time was 90 min. Translation products were analyzed by PAGE, immunoblotting, or trichloroacetic acid (TCA)–
precipitable counts (Mans and Novelli, 1961). Mitochondrial RNA
was translated using the reticulocyte lysate system (Promega).
Protein Gel Electrophoresis, Protein Gel Blotting,
and Immunodetection
Samples were run in an SDS-PAGE discontinuous system, as described by Laemmli (1970). Protein blotting was done using polyvinylidene difluoride membranes (Bio-Rad) in a tank system, as
described by Gallagher et al. (1995). Transfer buffer was 25 mM
Trizma base, 19 mM glycine, and 15% [v/v] methanol.
Immunodetection was conducted using the Vectastain ABC alkaline
phosphatase kit from Vector Laboratories (Burlingame, CA). Visualization was achieved by using 5-bromo-4-chloro-3-indolyl phosphate/
nitro blue tetrazolium substrates from Vector Laboratories.
Protein Overexpression and Antibody Production
Overexpression isolation and purification of Explon as a maltose
binding protein fusion were done following the manufacturer’s recommendations (New England Biolabs). After factor Xa cleavage, 100
mg of the purified AtLON peptide in complete Freund’s adjuvant was
injected into a rabbit, followed by a second dose 3 weeks later. After
three inoculation boosters, the antiserum was used to determine the
titer and specificity of the antibodies against the in vitro–translated
AtLON protease in protein gel blot assays. Preimmune serum was
taken before the first AtLON inoculation and used as a control in titrations of the anti-AtLON antibodies. The IgG fraction was purified
from anti-AtLON and anti-ORF239 antisera and preimmune sera by
using the Affi-Gel protein A MAPS II system from Bio-Rad. The
ORF239 antibody production was described previously (Abad et al.,
1995).
Linkage Analysis
In Organello Translations
A recombinant inbred family (Burr et al., 1988) between the common
bean Mesoamerican cultivar Jamapa and the Andean breeding line
Calima was used as the mapping population. This population con-
Mitochondrial translations were done as described by Forde et al.
(1978) without modifications. Total product of translation was used
1226
The Plant Cell
for immunoprecipitation of ORF239 products. In organello translations were performed with and without vanadate (4 mM), o-phenanthroline (5 mM), and N-ethylmaleimide (NEM) (2 mM).
Immunoprecipitations
After in organello translations, mitochondria were treated with digitonin and lubrol, as described by Ragan et al. (1987), without fractionation. Antiserum (6 mL) was added before incubation for 1 hr at room
temperature. Subsequently, 15 mL of Affi-Gel protein A–agarose
resin (Bio-Rad) was added to the mix and incubated for 1 hr at 48C.
Samples were centrifuged at 1500g for 1 min and washed twice with
binding buffer (Bio-Rad). Samples were resuspended in 20 mL of
Laemmli sample buffer (Laemmli, 1970) and boiled at 858C for 10
min. After a 15-sec centrifugation in an Eppendorf microcentrifuge,
supernatants were loaded onto the gel. Amplify (Amersham, Arlington Heights, IL) treatment of the gel was performed before gel drying
and exposure.
Proteolysis Assays
Resorufin-labeled casein (Boehringer Mannheim) was used in proteolysis assays. The labeled casein (90 mg) in 100 mM Tris-HCl, pH
7.5, 2 mM ATP, and 5 mM MgSO4 was used in each reaction for proteolysis. Sources of the LON protease were either 10 mL of in vitro
translation product or 10 mg of mitochondrial protein from inner
membrane or matrix fractions. Inhibitors used were NEM (2 mM),
vanadate (4 mM), o-phenanthroline (5 mM), and phenylmethylsulfonyl fluoride (PMSF) (4 mM) as well as apyrase (2 units). Incubations
were performed at 378C for 2 hr. Samples were taken every 30 min to
monitor degradation reactions. Determination of proteolytic activity
was done as recommended by Boehringer Mannheim. Absorbance
of the TCA-nonprecipitable peptides was measured in a Beckman
Instruments (Chicago, IL) DU640 spectrophotometer.
Proteolytic activity of the in vitro–translated lon clone and the inner
membrane and matrix fractions against the ORF239 substrate was
determined by adding the prepared extracts to an ORF239 in vitro
translation reaction. The in vitro–translated lon clone (10 mL) or 10 mg
of protein from a mitochondrial fraction was incubated with 25 mL of
TNT reticulocyte lysate (Promega), 2 mL of TNT buffer, 1 mL of an
amino acid mixture (1 mM) minus cysteine, 4 mL of 35S-cysteine at 10
mCi/mL (Amersham), 40 units of RNasin, 1 mL of T7 polymerase, 100
mM Tris-HCl, pH 7.5, 5 mM MgSO4, 2 mM ATP, and 1 mg of cloned
orf239 DNA. Inhibitors were used at concentrations indicated above,
and incubation time was 90 min, with samples being taken every 30
min. Post-translational turnover of ORF239 was monitored by incubating 10 mL of in vitro–translated Aralon or 10 mg of a mitochondrial
fraction with 5 mL of 35S-cysteine–labeled in vitro–translated
ORF239. The incubation was in the presence of 100 mM Tris-HCl, pH
7.5, 5 mM MgSO4, and 2 mM ATP. Inhibitor concentrations were as
indicated above. Reactions were sampled every 30 min for a total incubation time of 90 min.
To conduct assays for cotranslational turnover of the ORF239 substrate, it was necessary to demonstrate that the mitochondrial inner
membrane protease activity did not target components of the reticulocyte lysate necessary for the translation of ORF239. A control experiment was run in which the transcription/translation reaction mix
was preincubated with the inner membrane mitochondrial fraction
for 60 min at 308C in the absence of the Pvs-orf239 clone and the
protease inhibitor PMSF (4 mM). The treatment minus the orf239
clone and PMSF was subsequently incubated for 90 min at 308C in
the presence of the orf239 clone and PMSF (4 mM) and then analyzed by PAGE and autoradiography to confirm accumulation of the
35S-cysteine–labeled ORF239 peptide and little or no proteolytic activity. A second control reaction was performed as described above,
except that the mitochondrial fraction was omitted; this produced an
equal accumulation of the ORF239 peptide. In a third treatment, the
orf239 clone was added at the start of the incubation, but no PMSF
was included during the incubation to confirm that proteolytic activity
was present in the mitochondrial fractions used. As expected, the
amount of 35S-cysteine–labeled ORF239 accumulated in this treatment was considerably lower and proportional to what is presented
in Table 1.
In all proteolysis assays that involved the addition of anti-AtLON
antibodies or preimmune serum, 3 mL of purified IgG fraction was
used for incubation at room temperature with LON-containing extracts 30 min before the initiation of the proteolysis reactions. In
competition experiments, the in vitro–transcribed/translated AtLON
product was warmed to 658C for 5 min before the addition of the antibody. In all assays using 35S-cysteine–labeled ORF239, TCA-precipitable counts were determined using the procedure by Mans and
Novelli (1961). Duplicated samples for every time point were included
and read in an LS6500 Beckman scintillation counter.
Proteolysis data with the respective standard errors were plotted using the Sigmaplot program from Jandel Corporation (San Rafael, CA).
ACKNOWLEDGMENTS
We gratefully acknowledge Dr. Andre Abad for his important contributions at the beginning of this study and Dr. Mark Hermodson for
his critical review of the manuscript. We also thank Ms. Joanne
Cusumano and Dr. Clinton Chapple for their technical assistance and
prepared Arabidopsis materials for RNA gel blot analysis. Antibody
production was conducted through the Purdue Cancer Center Antibody Production Facility, and we thank John Wilder for his
assistance. This work was supported by a National Science Foundation grant (No. 9630252-MCB) to S.A.M.
Received December 29, 1997; accepted April 16, 1998.
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A Cytoplasmic Male Sterility−Associated Mitochondrial Peptide in Common Bean Is
Post-Translationally Regulated
Rodrigo Sarria, Anna Lyznik, C. Eduardo Vallejos and Sally A. Mackenzie
Plant Cell 1998;10;1217-1228
DOI 10.1105/tpc.10.7.1217
This information is current as of June 18, 2017
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