Plant Cell Physiol. 48(4): 655–661 (2007)
doi:10.1093/pcp/pcm031, available online at www.pcp.oxfordjournals.org
ß The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Short Communication
Uncovering a Link between a Plastid Translocon Component and Rhomboid
Proteases Using Yeast Mitochondria-Based Assays
Katherine Karakasis, Darcie Taylor and Kenton Ko *
Department of Biology, Queen’s University, Kingston, Ontario, Canada K7L 3N6
related processes. In Providencia stuartii, rhomboid-like
proteases are part of intercellular quorum sensing
(Gallio et al. 2002), and in Saccharomyces cerevisiae they
are involved in mitochondrial membrane remodeling. The
substrates identified for the yeast mitochondrial rhomboid
protease (YGR101w or Rbd1 or Pcp1) are cytochrome c
peroxidase (Ccp1) and a dynamin-like GTPase (Mgm1)
(McQuibban et al. 2003). There are also cases that show
limited or no substrate interchangeability, such as for some
mammalian rhomboid-like proteases (Lohi et al. 2004).
Rhomboid-like protein genes also exist in higher
plants. In Arabidopsis thaliana, there are at least eight
such genes predicted (using algorithms such as BLAST)
(Koonin et al. 2003). The encoded rhomboid-like proteins
are thought to be distributed throughout the plant cell,
including plastids and mitochondria (Koonin et al. 2003,
Kanaoka et al. 2005). Despite divergence from the
D. melanogaster sequence, one plant rhomboid protease,
AtRBL2, was shown to act similarly to the established
rhomboid proteases. In the mammalian cell co-transfection
assay system, AtRBL2 possesses the ability to cleave Spitz
and Keren, but not other known substrates (Kanaoka et al.
2005). This evidence indicates that plants contain active
rhomboid proteases and regulated intramembrane proteolysis (RIP). However, despite the presence of rhomboid-like
protease genes, A. thaliana does not appear to possess any
obvious counterparts to the substrates identified for the
D. melanogaster EGF signaling pathway (Kanaoka et al.
2005). Substrates or functional links for the plant rhomboid-like proteases are to date unknown, hence there are no
clues as to the biological roles of rhomboid proteases in
plants. In this study, a yeast mitochondria-based approach
was used to help uncover evidence of a possible link
between the plastid translocon component Tic40 and
conserved rhomboid proteases, thereby identifying plastid
protein transport as one potential cellular process in plants
to involve regulated intramembrane proteolysis.
Rhomboid proteins are site-specific proteases that
cleave substrates within the vicinity of a transmembrane
domain (TMD) (Weihofen and Martoglio 2003).
The soluble cleavage product is then released from the
Rhomboid proteases are present in bacteria, insects,
yeasts, parasites, mammals and plants. These proteases are
part of the regulated intramembrane proteolysis mechanism
for controlling processes such as development, stress response,
lipid metabolism and mitochondrial membrane remodeling.
Specific rhomboid protease substrates linked to these
processes have been identified from insects to mammals, but
not for plants. Identification of a link is a key step for
elucidating the role of each rhomboid protease. Here, using a
yeast mitochondria-based approach, we report evidence of a
potential link between a plastid translocon component and
organellar rhomboid proteases. This identification expands
the types of processes involving regulated intramembrane
proteolysis potentially to include at least one aspect of plastid
protein transport.
Keywords: Arabidopsis — Mutants — Plastid translocon —
Rhomboid proteases — Yeast mitochondria.
Abbreviations: AtRBL2, Arabidopsis rhomboid protease;
Ccp1, yeast mitochondrial cytochrome c peroxidase; Mgm1,
a yeast mitochondrial dynamin-like GTPase; Rbd1 and Rbd2,
yeast mitochondrial rhomboid proteases; RIP, regulated intramembrane proteolysis; Tic40, 40 kDa component of the translocon
of the inner chloroplast envelope; TMD, transmembrane domain.
Rhomboid serine proteases are structurally conserved
intramembrane enzymes. This conservation spans both
prokaryotic and eukaryotic kingdoms (Brown et al. 2000,
Koonin et al. 2003, Freeman 2006, Urban 2006, Wang et al.
2006). In many cases, these proteases show strong substrate
specificities, even across species. Drosophila melanogaster,
human and bacterial rhomboid proteases are able to cleave
specifically the same substrates, namely the D. melanogaster
epidermal growth factor (EGF) receptor ligands Spitz,
Keren and Gurken (Urban et al. 2001). These substrates
and their cleavage are part of the D. melanogaster
EGF signaling pathway. The activities of rhomboid
proteases are not, however, limited to such pathways.
Studies on bacterial and yeast rhomboid proteases
have revealed links to other types of targets and their
*Corresponding author: E-mail, [email protected]; Fax, þ1-613-533-6617.
655
656
A link between a plastid translocon component and rhomboid proteases
membrane and the other portion is secreted. Despite
substrate sequence diversity, the substrates identified so
far possess two features: solubility and a single predicted
TMD (Weihofen and Martoglio 2003). We have noticed
that the plastid translocon component Tic40 contains
similar features, a single predicted TMD near the
N-terminus (predicted using various algorithms) and a
soluble C-terminal segment (see hydropathy plots and
structural depictions in Fig. 1A). This similarity is evident
when Tic40 is compared with the two yeast mitochondrial
rhomboid protease substrates, Ccp1 and Mgm1. Moreover,
Tic40 is often observed as multiple immunoreactive protein
bands in blots of plastid samples, and possesses the ability
to associate with different sites in the plastid envelope
(Ko et al. 1995, Stahl et al. 1999, Chou et al. 2003). These
latter two properties of Tic40 also resemble the Ccp1–
Mgm1 situation in yeast mitochondria (Herlan et al. 2003,
Satoh et al. 2003, Sesaki et al. 2003, Herlan et al. 2004,
Michaelis et al. 2005, Sesaki et al. 2006). We therefore
assessed the possibility of a link between Tic40 and
rhomboid proteases by expressing full-length Ricinus
communis (castor bean plant) Tic40 in the same yeast
strains used in the Ccp1 and Mgm1 in vivo cleavage assays
(McQuibban et al. 2003) (Fig. 1B). Note that, at this stage,
the yeast mitochondria-based approach assesses direct
and/or indirect links. Potential links may manifest directly
as a substrate and/or indirectly through rhomboid proteaseregulated importation events. The strains used in our study
were: Invsc [as a non-mutated, diploid control strain and for
the purpose of comparing with previously observed patterns
(Ko et al. 2005)]; two homozygous rhomboid protease
mutants, rbd1D (YGR101w—lacks the identified mitochondrial rhomboid protease Rbd1) and rbd2D [YPL246c—a
predicted rhomboid protease mutant (Rbd2) with no known
phenotype and used as another control]; and a heterozygous
strain rbd1D/þ. Tic40 was expressed in all strains and was
localized to mitochondria, with no detectable levels in the
corresponding aqueous lysates. These results are in agreement with previous work showing the importation of Tic40
into mitochondria (Ko et al. 2005), as well as with the
targeting predictions arising from MitoProt and other
algorithms (Schwacke et al. 2004). The purity of the
mitochondrial fractions was confirmed using antibodies
against the matrix protein Arg8 and cytosolic hexokinase
(Ko et al. 2005; Fig. 1B). Similar band patterns of Tic40
(48, 44 and 42 kDa) were present in mitochondria isolated
from three of the four yeast strains, Invsc, rbd2D and the
heterozygous rbd1D/þ. This particular pattern appears
to be dependent on Rbd1 activity, since Tic40
exists predominantly as a 48 kDa band in the rbd1D cells.
There appears to be residual rhomboid protease
activity in the rbd1D strains, resulting in relatively low
amounts of similarly sized products (44 and 42 kDa).
Residual rhomboid protease activity has been observed in
other studies (e.g. McQuibban et al. 2003) and may be due
to Rbd2 in the system. The heterozygous rbd1D/þ strain
showed rescue of the defect exhibited in the homozygous
rbd1D strain. The multiple Tic40 band pattern (48, 44 and
42 kDa) reappeared in rbd1D/þ. Control mitochondrial
extracts prepared from yeast strains carrying plasmid alone
did not display immunoreactive bands (data shown for the
Invsc strain, Fig. 1B). Immunological specificity of the antiTic40 antibodies was established as described in Materials
and Methods and in previous studies (Ko et al. 1995,
Ko et al. 2005) (see Fig. 1B). Pre-immune antibodies do not
give rise to immunoreactive bands in any of the extracts—
yeast mitochondria, affinity-purified recombinant Tic40 or
plastids. Tic40 from A. thaliana (AtTic40 in Invsc) behaved
like the R. communis Tic40 (RcTic40), albeit with differences in the relative mobility of the bands. The more
obvious differences between the relative mobility of the
RcTic40 bands were technically advantageous and were
Fig. 1 Tic40 proteins incorporated into yeast mitochondria were examined immunologically. (A) A comparison of Tic40 hydropathy profiles
for Arabidopsis thaliana (AtTic40), Ricinus communis (RcTic40) and Pisum sativum (PsTic40) (using ExPaSy). The structure of Tic40 is
depicted (TP, transit peptide; TMD, transmembrane domain; TPR, tetratricopeptide repeats; J-domain, heat-shock protein-interacting domain
of co-chaperones). A potential import-processing site is indicated with a hatched vertical line. (B) Mitochondria or plastids from the indicated
sources were assessed with anti-Tic40 antibodies. The Invsc yeast samples are: Con, host Invsc extract (containing vector only); Rc AL,
aqueous lysate (post-mitochondrial fraction) of RcTic40-producing cells; Rc mito, mitochondrial fraction corresponding to Rc AL; At mito,
mitochondria from AtTic40-producing cells. Mitochondrial and aqueous subfractions were assessed immunologically for the matrix protein
Arg8 and cytosolic hexokinase. Mitochondrial samples under the three mutant yeast strains all contain RcTic40 (Rc mito). These
mitochondrial samples were also assessed for Arg8 as one type of control experiment. Plastid extracts are from R. communis (Rc), transgenic A.
thaliana producing RcTic40 (Trans At) and wild-type A. thaliana (Wt At). Relative masses (kDa) are given at the far left. Antibody specificity is
shown in the lower right of (B). (C) The same rbd1D and rbd2D samples from (B) were assessed for Tom40, Tim44, mtHsp70 and Arg8 (matrix
protein). (D) Mitochondria isolated from the indicated yeast strains were given a mock reaction (Con), thermolysin (Therm) or trypsin (Tryp) to
assess the physical status of Tic40. Smaller, trypsin-generated products are noted with asterisks. Note that the horizontal black lines delineate
sections of images that required adjustments to contrast. Samples were also assessed for Arg8 as one type of control experiment. Protease
control experiments (thermolysin or trypsin) were conducted in the presence of detergent (Triton X-100) and assessed immunologically as
above. Equal amounts of total mitochondrial or plastidial protein were used for each of the samples assessed in the various experiments
presented. Details for the anti-Tic40 antibodies employed are given in Materials and Methods. Groupings of images of different exposures
from different blots (B) or different portions of simultaneously run blots (D) are delineated by black lines.
A link between a plastid translocon component and rhomboid proteases
657
A
Arabidopsis Tic40
Ricinus Tic40
CH3
Pisum Tic40
CH3
4.0
3.0
2.0
1.0
0.0
−1.0
−2.0
−3.0
−4.0
H2O
50
CH3
4.0
3.0
2.0
1.0
0.0
−1.0
−2.0
−3.0
−4.0
H2O
450
50
4.0
3.0
2.0
1.0
0.0
−1.0
−2.0
−3.0
−4.0
H2O
450
50
450
SOLUBLE SEGMENT
N
TP REGION
TMD
C
TPR
J-DOMAIN
Potential locations of cleavage site(s)
1
87
114 134
254
398
460
Amino acid position
B
Yeast
Yeast
rbd1∆
Invsc
48 kDa
44 kDa
42 kDa
rbd2∆
Rc AL Rc mito At mito
Subfractionation markers
Plastid extracts
48 kDa
48 kDa
44 kDa
42 kDa
Con
Plants
rbd1∆ /
rbd1
42 kDa
Rc mito Rc mito Rc mito
Rc
Trans At Wt At
Antibodies used
Arg8
Arg8
Hexokinase
AL
Preimmune Recombinant
Serum
Tic40
Mito
C
Tom40
rbd1∆
rbd2∆
Tim44
rbd1∆
mtHsp70
rbd2∆
rbd1∆
rbd2∆
Arg8
rbd1∆
rbd2∆
D
rbd1∆
Invsc
rbd2∆
Protease control
48 kDa
44 kDa
42 kDa
Tic40
Arg8
Con
Arg8
Fig. 1
Therm
Tryp
Con
Therm
Tryp
Con
Therm
Tryp
658
A link between a plastid translocon component and rhomboid proteases
utilized for most of the experiments. The bands that appear
in yeast mitochondria are also detectable in R. communis
plastid extracts (Fig. 1B), suggesting that plastids may
contain similar rhomboid protease-based activities.
Furthermore, plastids from transgenic A. thaliana plants
co-producing RcTic40 show a pattern similar to that of
R. communis plastid extracts. Control A. thaliana plastid
extracts (Fig. 1B) did not exhibit such a distinct pattern.
Differences in band intensity between the systems studied
probably reflect variations in rhomboid protease activities.
Such differences have been observed in other studies,
e.g. during substrate cleavage (Urban et al. 2001).
Also, the apparent differences in the pattern of the 44 and
42 kDa Tic40 forms observed are due to their high
levels in the sampled plant tissues. As a result, these two
forms migrate as one wider intense band in protein gels
(unpublished 2D gel data).
To determine if there were any obvious defects in the
yeast mitochondrial translocon that may have contributed
to the Tic40 band patterns, the same rbd1D and rbd2D
mitochondria samples used in Fig. 1B were further analyzed
with antibodies against various yeast mitochondrial
translocon components. We assessed the samples for
13 different components and found no obvious evidence
that the transport machinery was structurally impaired
(data shown for three of the 13 components tested). Profiles
for the mitochondrial translocon components Tom40,
Tim44 and matrix heat-shock protein 70 are shown in
Fig. 1C (translocon of the outer and inner membrane,
respectively). The comparable steady-state levels of the
matrix protein Arg8 observed for the yeast strains suggest
that protein accumulation was not affected in an obvious
manner. These profiles also acted as control immunoblots
for the experiments presented in Fig. 1B.
To assess whether the observed Tic40 patterns were
due to arrest along the transport route, isolated mitochondria from each of the three Tic40-expressing yeast strains
(Invsc, rbd1D and rbd2D) were treated with exogenously
added proteases (thermolysin or trypsin). Thermolysin
is accessible only to surface-exposed proteins, whereas
trypsin is also accessible to proteins internal to the outer
membrane. The results indicate that most of the Tic40
proteins were imported and were fully or partly protected
from exogenously added proteases (Fig. 1D). Focusing
mainly on the largest Tic40 form, both thermolysin and
trypsin were able to cleave the 48 kDa band in Invsc and
rbd2D, indicating that this form was at least accessible from
the cytosolic side of the outer membrane. Interestingly, the
Tic40 proteins in rbd1D, including the largest one, were
largely protected from both proteases. Most of the largest
Tic40 form in rbd1D did not appear to be from arresting
translocation at an early stage and becoming external to the
organelle as a consequence. Trypsin treatment gave rise to
low amounts of smaller Tic40 bands in all three cases
(marked by asterisks in Fig. 1D). The matrix protein Arg8
remained protected for all treatments (Fig. 1D). Tic40
proteins in yeast mitochondria were degraded by thermolysin or trypsin in the presence of detergent (Fig. 1D,
protease controls), as shown previously for plastid samples
(Ko et al. 1995). The protease pattern displayed by rbd1D
suggests that rhomboid protease loss may create changes to
cleavage and Tic40 translocation. This aspect is currently
under investigation. The results for Invsc and rbd2D are in
line with the protease accessibility patterns previously
observed for plastidial Tic40, in plants and in plastid
import assays (Fig. 1B, and in Ko et al. 1995, Ko and
Ko 1999, Ko et al. 2005). This correlation implies that
importation of Tic40 in yeast mitochondria is similar to
that in plastids and that the observed Tic40 patterns are
probably due to the involvement of the yeast mitochondrial
rhomboid protease.
Since events related to the process of importation are
involved, we needed to determine if activities resembling
those of rhomboid proteases were responsible for the
generation of the Tic40 band patterns. The behavior of
rhomboid proteases is distinct, recognizing and cleaving
specific sites in a TMD region. Various deletions of Tic40
were thus constructed to see if the cleavage pattern of
Tic40 resembles that of other established rhomboid
protease substrates and to assess the directness of the
Tic40–rhomboid protease link. All deletions tested contain
sufficient amounts of primary targeting information at
the N-terminus to allow passage into mitochondria and
exposure to rhomboid proteases in the inner membrane.
Four of the deletions tested were in the N-terminal segment
immediately upstream of the predicted TMD region
(the ‘N-terminal’ deletions) and the two others were
downstream in the soluble portion (the ‘C-terminal’
deletions) (Fig. 2). The deletions were designed strategically
to see if cleavage occurred in the predicted Tic40 TMD
region and were assayed in vivo, as above, using rbd1D
and rbd2D cells (Fig. 2). TMD deletion resulted in the
re-direction of Tic40 to the matrix and was not assayed
further. Again, the six deletion constructs behaved in a
manner similar to that of unaltered Tic40. Like unaltered
Tic40, residual activity was also observed in these experiments. Despite the size differences and number of bands
observed as a function of the deletions, mitochondria
containing the four ‘N-terminal’ deletions displayed at least
one common sized band (42 kDa) among the different sized
forms. Cleavage of the two ‘C-terminal’ deletions appeared
largely unaffected, despite the lower levels of the residual
activity observed relative to Tic40. Collectively, the patterns
obtained with the six deletion constructs help delineate one
segment where Tic40 shows possible cleavage by Rbd1 or
an Rbd1-regulated protease. Cleavage at this site appears to
A link between a plastid translocon component and rhomboid proteases
Deletion
43
Tic40
in
rbd1∆
115
1
TP
2
TP
3
TP
4
TP
TMD
5
TP
TMD
TMD
74
A
115
TMD
TMD
Tic40 &
Tic40 &
At1g74130 pACT2 with
in control DNA insert
rbd1∆
in rbd1∆
Tic40
in
rbd2∆
N-terminal
Deletions
88 115
659
Glucose
43 74
143
218
217
6
TP
TMD
N-terminal Tic40 deletions
Deletion 2
Deletion 1
rbd1∆ rbd2∆
rbd1∆ rbd2∆
Arg8
rbd1∆ rbd2∆
C-terminal Tic40 deletions
Deletion 6
Deletion 5
35 kDa
32 kDa
30 kDa
rbd1∆ rbd2∆
pACT2 with
control DNA At1g74130
insert
Deletion 4
46 kDa
42 kDa
39 kDa
37 kDa
35 kDa
Plant rhomboid
protease in rbd1∆
rbd1∆ rbd2∆
Deletion 3
44 kDa
42 kDa
40 kDa
B
48 kDa
44 kDa
42 kDa
44 kDa
42 kDa
40 kDa
42 kDa
40 kDa
Glycerol
C-terminal
Deletions
315
rbd1∆ rbd2∆
Fig. 2 Assessing the cleavage pattern of different Tic40 deletions
in yeast mitochondria. The various Tic40 deletions shown
diagrammatically were assayed in rbd1D and rbd2D yeast cells
as described in Fig. 1. Construction details for the deletions are
given in Materials and Methods. Immunoblot patterns are shown
for each Tic40 deletion. The arrowhead indicates the common
sized band for the N-terminal deletion set. Relative molecular
masses (kDa) are indicated. Equal amounts of total mitochondrial
protein were used for each of the samples assessed.
See Supplementary Fig. S1 for a comparison of relative molecular
masses and for control experiments.
occur in a 30 amino acid segment located in the predicted
TMD region. This segment is at least 28 amino acids from
the predicted import processing site, and is thus distinct
(Stahl et al. 1999). The presence of other bands indicates
that there may be other cleavage sites and/or sequences that
affect cleavage activity, made apparent by the deletions.
This aspect requires further study. Although importation is
involved, these results provide another line of evidence that
the appearance of multiple Tic40 bands (48, 44 and 42 kDa)
is linked to the activities of the yeast mitochondrial
rhomboid protease.
The levels of amino acid similarity between the three
predicted Arabidopsis organellar rhomboid proteases
(At1g18600, At1g25290 and At1g74130) and yeast mitochondrial Rbd1 are approximately 22–23% (Supplementary
Fig. S2). To determine if rescue of the yeast mutant rbd1D
Fig. 3 Assessing the link between Tic40 and a plant organellar
rhomboid protease in yeast. (A) Growth of various rbd1D and
rbd2D yeast strains on glucose- and glycerol-supplemented media.
The strains are either expressing Tic40 alone or in combination
with a select Arabidopsis thaliana rhomboid protease. (B) The
presence of At1g74130 plant rhomboid protease in rbd1D partially
restores the pattern of Tic40 bands characteristic of the one
observed in rbd2D. Equal amounts of total mitochondrial protein
were used for the two samples assessed. As in Figs. 1 and 2, the
samples were also assessed immunologically for Arg8 as a control.
Groupings of images from different portions of simultaneously run
blots (A) or different portions of the same blot or simultaneously run
blots (C) are delineated by black lines.
was possible and to see if the Tic40 pattern reverted to the
one characterized for rbd2D, we utilized the above rbd1D
in vivo assay to assess one of the predicted rhomboid
proteases (At1g74130). The constitutive expression of
At1g74130 displayed a limited ability to rescue the growth
of rbd1D in glycerol-supplemented media (Fig. 3A).
The strain containing pACT2 with a control DNA insert
did not exhibit rescue capabilities. The Tic40 band pattern
observed for rbd2D was partially restored in rbd1D by the
presence of At1g74130 (Fig. 3B). Again, control cells did
not display any restoration of the Tic40 band pattern.
The ability to restore only partially appears to reflect the
level of rhomboid protease activity displayed in plants
(data to be reported in a future study). Also, the
observations so far obtained with Arabidopsis T-DNA
insertion mutants of these proteases provide more indicators of a link between Tic40 and plant organellar rhomboid
proteases (data to be reported in a future study).
These results together provide more evidence that Tic40’s
protein pattern is linked to the activities of conserved
rhomboid proteases and that the link is likely to operate in
plants as well.
660
A link between a plastid translocon component and rhomboid proteases
Regulated intramembrane proteolysis is a new paradigm
in cell biology and is emerging as a way to influence specific
cellular processes. Inclusion of the rhomboid protease family
in the RIP phenomenon is a recent advancement, especially
for mitochondrial rhomboid proteases. It is now becoming
clear that RIP is one general mechanism for releasing
functional protein domains from their transmembrane
anchors. The current evidence for yeast Rbd1 suggests two
related roles for this rhomboid protease, one for Ccp1 and
Mgm1 maturation, and the other for regulating the generation of different substrate forms to meet cellular needs.
Mgm1 exemplifies this phenomenon, where long and short
forms possess functionality (Herlan et al. 2003, Satoh et al.
2003, Sesaki et al. 2003, Herlan et al. 2004, Sesaki et al. 2006).
The Tic40 situation in plastids may operate similarly. Clues
for this can be found in previous work on Tic40, such as the
existence of different configurations, modulating capabilities
and preferential protein–protein interactions (Ko et al. 1995,
Ko et al. 2004, Ko et al. 2005). Tic40 is often observed as
more than one immunoreactive protein band in blots of
plastid envelope samples and possesses the ability to associate
with different sites in the plastid envelope such as Toc75 and
Tic110 (Ko et al. 1995, Stahl et al. 1999, Chou et al. 2003).
More recently, an intermediate Tic40 form was observed in
inner envelope vesicles as part of the internal targeting
pathway after importation (Li and Schnell 2006). The
derivation of this intermediate Tic40 form appears to involve
a signal-processing peptidase and another unidentified
processing factor. The cleavage events reported in this
study may possibly be related to the unidentified processing
protease, but this needs confirmation. At this point, it is
tempting to speculate that there may be a possible link
between the different Tic40 forms and the rhomboid protease
activities observed here, but this speculation also needs
further investigation. Using experimental strategies built on
the universal nature of rhomboid proteases (Brown et al.
2000), we have shown here that there is a potential
relationship between the activities of Tic40 and organellar
rhomboid proteases. The relationship can be direct and/or
indirect in nature. The combination of findings suggests that
rhomboid proteases are involved not only in Tic40 maturation, but possibly in its modulatory capabilities. Since the
biological roles of plant rhomboid proteases and RIP have
yet to be elucidated, the identification of a possible link
between Tic40 and organellar rhomboid proteases sets the
stage for investigating the potential relationship between
rhomboid proteases, RIP and plant cellular processes such as
plastid protein transport. The presence of multiple genes for
organellar rhomboid proteases suggests that the Tic40–
rhomboid protease link is likely to be complex and will
need to be investigated with this in mind. Hence, the
possibility of rhomboid proteases with different specificities
and activities working together needs to be considered.
The location and physiological significance of rhomboid
proteases in plants, and the possibility that Tic40 is a
rhomboid protease substrate itself are currently being
elucidated. These findings will be reported in the near future.
Materials and Methods
The production of R. communis and A. thaliana Tic40
(accession Nos. DQ473580 and AY093010, respectively) in yeast
was achieved by inserting the corresponding cDNA fragments into
the yeast–Escherichia coli shuttle vector YEplac195 (Gietz and
Sugino, 1988). The production of plant rhomboid proteases
(At1g18600 or At1g74130) in yeast was carried out in the same
manner as above using pACT2 (Clontech Laboratories, Mountain
View, CA, USA). Expression was achieved using the alcohol
dehydrogenase (ADH) promoter. Plasmids were introduced into
the indicated yeast strains using standard yeast transformation
techniques. The Invsc yeast strain was purchased from Invitrogen.
The three mutant strains were purchased from EUROSCARF
(homozygous rbd1D, homozygous rbd2D and heterozygous
rbd1D/þ). All strains were grown in glucose-supplemented
medium. The isolation of mitochondria was conducted as
described (Daum et al. 1982). Treatment of mitochondria with
the proteases thermolysin and trypsin was carried out according to
Yamaguchi and Hatefi (1991) and Jascur (1991), respectively.
All protein samples were quantitated and normalized before
immunological assessment. Standard immunoblotting techniques
were used. The rabbit polyclonal anti-Tic40 antibodies used were
equally immunoreactive for all Tic40 forms, independent of
derivation source (yeast, bacteria or plant species) (Ko et al.
1995). These antibodies were made against recombinant proteins
encompassing the soluble stromal portion of Tic40 (without the
TMD region) (Ko et al. 1995). Immunological specificity of the
anti-Tic40 antibodies was confirmed by control immunoblot
experiments using rabbit pre-immune serum and inherently
through immunoblots employing other specific antibodies
(Fig. 1B, C). Immunological specificity was further confirmed
using affinity-purified antibodies (data not shown) or affinitypurified recombinant Tic40 (reported in Ko et al. 2004)
(see Fig. 1B). Control yeast mitochondrial extracts (from yeast
strains carrying plasmid only) were also assessed with the antiTic40 antibodies to confirm specificity, i.e. no immunoreacting
bands observed in the corresponding protein blots (Fig. 1B).
The 1 : 1,000 dilution of anti-Tic40 antibodies used was determined
by titration and linearity experiments. All immunoblot experiments
were repeated at least three times, and representative results are
shown in the different figures. The production of deletion proteins
in yeast was achieved in the same manner as above. Deletions 1–6
were generated by removing the corresponding DNA sequence
from the Tic40 cDNA fragment as follows: (i) deletion of 72
residues by removing a 216 bp SacI–BstEII fragment; (ii) deletion
of 41 residues by removing a 123 bp HpaII–BstEII fragment;
(iii) deletion of 27 residues by removing an 81 bp TaqI–BstEII
fragment; (iv) deletion of 31 residues by removing a 93 bp
SacI–HpaII fragment; (v) deletion of 75 residues by removing a
225 bp HindIII–PstI fragment; and (vi) Deletion of 98 residues by
removing a 294 bp PstI–HindIII fragment.
Supplementary material
Supplementary material mentioned in the article is available to
online subscribers at the journal website www.pcp.oxfordjournals.org.
A link between a plastid translocon component and rhomboid proteases
Acknowledgments
We thank C. Boone for providing the plasmid p195,
EUROSCARF for yeast mutant strains, RIKEN for At1g74130
cDNA, and generous colleagues for IgGs against various yeast
mitochondrial proteins: E. Craig for mtHsp70; N. Pfanner for
Tim50, Tom22, 40 and 70; T. Endo for Tim50; C. Koehler for
Tim22 and Tim44; R. Jensen for Tim10, 17, 18, 23 and 54; and
T. Fox for Arg8. The work was supported by a grant from the
Natural Sciences and Engineering Research Council of Canada.
The authors thank Drs. I. Chin-Sang, P. Davies and V. Walker for
critiquing the manuscript.
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(Received January 9, 2007; Accepted February 21, 2007)