(Received for publication, August 28, 1995)
From the
Many of the proteins in the chloroplast envelope play an important role in facilitating the biochemical and transport processes of the compartment. For the transport of proteins into the chloroplast, we have recently identified at least three different envelope proteins (Com44/Cim44, Com70, and Cim97) in close physical proximity to a partially translocated chimeric precursor protein (Wu, C., Seibert, F. S., and Ko, K.(1994) J. Biol. Chem. 269, 32264-32271). In this study we report the characterization of a cDNA clone encoding a member of the Com44/Cim44 envelope proteins. The combined data from nucleotide sequencing, and RNA and protein blot analyses indicate the existence of multiple forms of the 44-kDa envelope protein. Depending on the plant species examined, immunologically-related protein bands with molecular masses of 42 to 46 kDa were observed. Organelle subfractionation, protease treatment, and immunomicroscopic studies together provide an indication that the immunologically-related proteins may be present in both the outer and inner envelope membranes. Co-migration of the product synthesized from the cDNA insert with a 44-kDa immunoreactive band of the chloroplast envelope, and the in vitro import results, together suggest that the in vitro synthesized 44-kDa protein is targeted to the envelope membrane without any further processing.
Chloroplast envelope proteins play a major role in modulating the vectorial flow of molecules across the membrane, including large proteinaceous entities. The import of proteins into the chloroplast is a complex process requiring the close collaboration of both the outer envelope and the inner envelope membranes. Evidence for the possible existence of two distinct protein import complexes, one in each envelope membrane, is beginning to emerge from a number of recent investigations (Waegemann and Soll, 1991; Soll and Waegemann, 1992; Schnell and Blobel, 1993; Alefson et al., 1994; Schell et al., 1994; Kessler et al., 1994; Wu et al., 1994). An important step in the characterization of the protein translocating complexes is the identification of the components involved. The identification of outer and inner envelope polypeptides of these protein translocating complexes has been achieved using a variety of strategies (Cornwall and Keegstra, 1987; Kaderbhai et al., 1988; Pain et al., 1988; Schnell et al., 1990a, 1994; Hinz and Flugge, 1988; Soll and Waegemann, 1992; Waegemann et al., 1990; Perry and Keegstra, 1994; Alefson et al., 1994; Kessler et al., 1994; Wu et al., 1994; Hirsch et al., 1994; Seedorf et al., 1995; Seedorf and Soll, 1995; Gray and Row, 1995). So far these studies collectively indicate that envelope proteins with molecular masses of 30, 34, 36, 44, 45, 51, 66, 70, 75, 86, 97, and 100 kDa may be possible constituents of the chloroplast protein import apparatus; however, it is not obvious from the existing data whether some of the predicted similar sized components are identical to each other.
The complex nature of protein translocation mechanisms observed in other membranous systems, such as the mitochondrion and the endoplasmic reticulum, suggests that there is most likely a significant number of chloroplast envelope components that need to be identified and characterized in detail. Our major strategy for identifying and studying components of the protein translocation apparatus is to isolate cDNA clones that encode all types of chloroplast envelope proteins and then to systematically sort out the identity and/or function of the clones. This approach allows us to circumvent the technical problems and limitations of purifying small quantities of authentic proteins from the envelope. In this study, we report on the identification and molecular characterization of one of the cDNA clones that encodes a 44-kDa envelope protein with unusual features. The 44-kDa polypeptide encoded by this cDNA insert is a member of the Com44/Cim44 chloroplast envelope proteins recently found in close physical proximity to a partially translocated chimeric precursor protein (Wu et al., 1994). Specific antibodies raised against the 44-kDa protein were used to determine the location of the immunologically-related polypeptides in the chloroplast envelope. The implications of the potential locale of these immunologically-related proteins are discussed in relation to recent developments in our understanding of the uptake of proteins into the chloroplast.
Immunoelectron microscopy was performed according to Pain et al.(1988). Sections of treated intact chloroplasts were examined using a Zeiss CR-10 electron microscope.
Figure 1: Nucleotide and deduced amino acid sequences of the cDNA clones encoding immunologically-related 44-kDa envelope proteins. DNA sequences and corresponding deduced protein sequences are compared. Sequence information is presented for both Brassica cDNA clones, bce44A (partial length) (lines designated A) and bce44B (full-length) (lines designated by B). Nucleotide positions are indicated on the right side. The beginning of the 3`-untranslated region of the DNA sequences is marked by a solid arrowhead.
Antibodies against the cDNA-encoded Bce44B
(NH and COOH terminus) gave rise to identical chemical
cross-linking/immunoprecipitation results as reported by Wu et
al.(1994) (Fig. 2), providing a confirmation of
Bce44B's identity and its predicted function. The membrane
impermeable chemical cross-linker DTSSP allowed the formation of
cross-linked complexes between Bce44B and partially translocated
Oee1-Dhfr precursors (Fig. 2, lane 1). These complexes
were immunoprecipitated only with antibodies against Bce44B but not the
abundant 37-kDa inner membrane protein (Fig. 2, lane
4). Cross-linked complexes were not immunoprecipitated with
preimmune IgGs (Fig. 2, lane 5) or when the assays were
conducted with 1 mM ATP (Fig. 2, lane 2) or in
the absence of DTSSP (Fig. 2, lane 3). The ability of
DTSSP to form cross-linked complexes indicates accessibility from the
cytosolic side of the envelope.
Figure 2: Cross-linking the translocating Oee1-Dhfr precursor to Bce44B. A control import assay conducted with 50 µM ATP is presented in lane 6. The precursor and the translocation intermediate are indicated by p and i*, respectively. The antibodies used for immunoprecipitation of the cross-linked complexes are as follows: anti-Bce44B (lanes 1-3), anti-37-kDa (lane 4), and preimmune (lane 5). Lanes 1, 2, 4, and 5 represent results in the presence of chemical cross-linker DTSSP. Lane 3 represent results in the absence of chemical cross-linking. Lane 2 represents an experiment conducted with 1 mM ATP instead of the 50 µM ATP used in other lanes.
The nucleotide sequence of the bce44B cDNA insert (sequences marked B in Fig. 1) was found to contain an open reading frame of 969 nucleotides plus 122 and 102 nucleotides of 5`- and 3`-untranslated sequences, respectively. The total length of the cDNA insert is 1,193 nucleotides, compared with an estimated mRNA size of 1,200 nucleotides (Fig. 3), suggesting that the cDNA insert is probably full-length. The initiating methionine codon was assigned to nucleotides 122-124 which results in a protein of 323 amino acids. The deduced protein sequence was calculated to have a molecular mass of 36 kDa which is substantially less than the value estimated by SDS-PAGE. This difference may be attributed to some undetermined structural feature of the protein that affects its migration in SDS-polyacrylamide gels. Similar discrepancies have been noted before, especially for proteins originating from membranous locations, e.g. Omp24 (Fisher et al., 1994). This possibility is supported by the fact that the in vitro product synthesized from the cDNA insert co-migrated with the native B. napus envelope protein (discussed in the import results). The hydropathy profile generated for the deduced bce44B protein sequence did not show any clear dominant hydrophobic or membrane-spanning features which could have given us a clue as to its location in the chloroplast envelope membrane (data not shown). This characteristic is reflected in the amino acid composition: 48.6% for nonpolar and 47.7% for polar residues. The amino-terminal 62 residues are completely devoid of acidic residues, a feature characteristic of targeting sequences. Comparison of the bce44AB nucleotide and deduced amino acid sequences to entries in the gene data banks did not reveal any identity/function at the time of manuscript preparation. The deduced BCE44B protein sequence is identical to the partial sequence for the tomato version of this polypeptide (Tce44) (data not shown), providing sequence confirmation that Bce44B is identical to the protein used to generate IgGs for the previous chemical cross-linking study (Wu et al., 1994).
Figure 3: RNA blot analysis of B. napus. RNA blot analysis of steady-state bce44A and bce44B transcript levels in response to light-dark treatments. Hybridization experiments were carried out using cDNA probes for bce44A (lanes 1 and 2) and bce44B (lanes 3 and 4). Total RNA from dark treated and light grown plants is indicated by D and L, respectively. A control experiment using the rbcS probe is also presented (second row marked RBCS). The estimated size of the bce44B transcript is indicated in nucleotides.
The cDNA sequence data indicate that the
transcripts (bce44A and bce44B) are likely derived
from different genes since the 3`-untranslated regions are different (Fig. 1). This possibility was confirmed by the distinct genomic
DNA hybridization patterns displayed by the two cDNA clones. ()Differences were also observed at the steady state
transcript level (Fig. 3). The amount of steady state bce44B transcripts was relatively low when compared to the highly
abundant transcripts of rbcS. The bce44B transcripts
were estimated to be approximately 1,200 nucleotides long. The steady
state mRNA levels of bce44B were not significantly influenced
by light. The bce44B mRNA level appears to remain constant
despite dark treatment for 3 days, whereas steady state mRNA levels of
the positive light-regulated rbcS gene decreased to a very low
amount. Steady state transcripts were not detected with the bce44A probe (Fig. 3, lanes 1 and 2) and were
not influenced by light such that the transcripts became detectable
after a dark treatment. These results indicate that bce44B possesses an expression profile different from bce44A,
further supporting the possibility that bce44A is distinct
from bce44B.
Figure 4: Immunoblot analysis of the 44-kDa chloroplast envelope proteins. A, a comparison of immunoreactive 44-kDa envelope bands in total chloroplast samples prepared from pea, tomato, and B. napus is presented in lanes 1-3, respectively. The anti-44-kDa IgGs were also reacted to total pea chloroplast proteins (lane 4) and total pea plant cell proteins (lane 5) that were extracted with 10% trichloroacetic acid. B, in the first row, anti-44-kDa IgGs were reacted to total stromal proteins (lane 1), total thylakoid proteins (lane 2), total envelope fraction (lane 3), outer (lane 4) and inner (lane 5) envelope fractions. For comparison, the same samples were reacted with anti-37 kDa (second row marked 37 kDa) and anti-Com70 IgGs (third row marked 70 kDa), representing inner and outer envelope proteins, respectively. The same fractions were also probed with preimmune IgGs (fourth row marked CON).
Thermolysin and trypsin treatments of pea chloroplasts were conducted to further examine the nature of the 44-kDa proteins' association with the envelope. Thermolysin is a protease that cannot penetrate the outer envelope hence it is useful for probing polypeptides accessible on the surface of the outer membrane. Trypsin can penetrate the outer envelope and can thus be used to probe accessible protein moieties external to the inner envelope membrane (Joyard et al., 1983; Cline et al., 1985). Identical results were obtained with whole organelles and with envelopes prepared from treated plastids as well as with B. napus chloroplasts, therefore only the results of the pea chloroplast envelope experiment are presented (Fig. 5A). The majority of the immunorelated 44-kDa proteins are resistant to proteases (both thermolysin and trypsin) and are probably protected by the inner envelope membrane. However, thermolysin treatment gave rise to small amounts of a polypeptide with a relative molecular mass of approximately 42 kDa (Fig. 5A, lane 2). The thermolysin-generated 42-kDa band was, however, degraded with a subsequent trypsin treatment or by using trypsin in place of thermolysin (Fig. 5A, lane 3). A distinct smaller sized trypsin-generated immunoreactive product of approximately 30 kDa was observed (Fig. 5A, lane 3). The thermolysin-generated 42-kDa band is distinct from the native 42-kDa immunoreactive protein since the native band is still present in the same amount after the trypsin treatment (Fig. 5A, lane 3), indicating that the native 42-kDa band is resistant to trypsin. The densitometer scans indicate that the decrease in the amount of native 44-kDa protein after a protease treatment was similar for the two types of proteases used, suggesting that the protease-sensitive 44-kDa form is present in a finite amount and is distinct from the protease-resistant 44-kDa forms. The protease degradation characteristics displayed by the protease-sensitive 44-kDa form suggests that this protein was accessible from the external side of the outer envelope. The 37-kDa inner envelope protein was unaffected by both types of proteases and was used as an internal control and for normalization purposes (Fig. 5A). All immunorelated 44-kDa forms were susceptible to proteases in the presence of 0.1% Triton X-100 (Fig. 5B).
Figure 5: Analysis of the 44-kDa proteins by protease treatment. A, the 44-kDa proteins were analyzed using pea total envelope fractions purified after the prescribed treatment scheme. Lanes 1-3 represent isolated mixed envelopes with no treatment, thermolysin- (100 µg/ml) and trypsin (75 µg/ml)-treated total envelopes, respectively. Chloroplast samples given the same treatments as in the first row were probed with anti-37-kDa IgGs in the second row marked 37 kDa. B, total pea envelopes were treated with 1% Triton X-100 and thermolysin (100 µg/ml) (lane 1) or trypsin (75 µg/ml) (lane 2). C, purified inner envelopes (equivalent to 10 µg of protein) were treated with thermolysin (100 µg/ml). Lanes 1 and 2 represent treatments with 0 and 100 µg/ml thermolysin, respectively. The samples were probed with anti-44-kDa (first row marked 44 kDa) and with anti-37 kDa IgGs (second row marked 37 kDa).
The major protease-resistant, immunorelated 44-kDa forms of the inner envelope were sensitive to thermolysin only after isolated inner membranes were given a post-fractionation thermolysin treatment (Fig. 5C). Thermolysin cleaved all inner envelope immunorelated 44-kDa forms into 42-kDa products, indicating that these proteins were partly protected by the inner membrane. The 37-kDa inner membrane protein also exhibited the same characteristics, giving rise to a 35-kDa band. Since treatment of chloroplasts with trypsin (Fig. 5C) did not give rise to a similar pattern of protease-degraded products as with fractionated inner envelopes, the evidence suggests that the thermolysin-sensitive 44-kDa form in the outer envelope is distinct from the inner membrane 44-kDa forms.
Two immunomicroscopic techniques were used to further examine the possibility of immunorelated 44-kDa proteins in the outer and inner envelope. Isolated intact chloroplasts were subjected to immunofluorescence labeling as described under ``Materials and Methods.'' Incubation of intact chloroplasts with buffer or preimmune IgGs followed by a secondary labeling reaction with fluorescein isothiocyanate-conjugated goat anti-rabbit IgGs did not result in fluorescent labeling of the plastids (Fig. 6, A and B). A primary incubation with anti-44-kDa antibodies yielded a patched pattern of immunofluorescence (Fig. 6C), indicating accessibility from the external side of the outer envelope.
Figure 6:
Localization of the 44-kDa proteins by
indirect immunofluorescence microscopy. Pea chloroplasts were incubated
with (A) buffer, (B) preimmune rabbit IgGs, or (C) anti-44-kDa rabbit IgGs. Bound antibodies were then
visualized with fluorescein isothiocyanate-conjugated goat anti-rabbit
IgGs. An example of the immunofluorescent patterns observed are shown
in C. The magnifications in panels A-C are
1416,
1416, and
1950,
respectively.
The immunoelectron microscopy results also suggest the same possibility as that found in the above experiments. Intact chloroplasts were decorated with gold in discrete areas of the envelope membrane (Fig. 7), a pattern similar to the one obtained by immunofluorescence microscopy. The majority of the labeling was observed to be in clusters suggesting that the antigenic moieties were accessible from the outside. Immunogold labeling was not observed with chloroplasts incubated with gold-conjugated IgGs alone or with preimmune IgGs (data not shown).
Figure 7:
Localization of the 44-kDa proteins in
intact pea chloroplasts by immunoelectron microscopy. Pea chloroplasts
were prepared and immunodecorated with gold-conjugated goat anti-rabbit
IgG. An example of immunogold labeling at the chloroplast surface at
various magnifications are presented. The arrows indicate the
positions of immunogold label on the chloroplast (49,200). A and B represent higher magnification of corresponding
regions marked by arrows. Magnifications are
103,400
and
110,000, respectively.
Figure 8: Import characteristics of Bce44B. The resulting fluorograms of total chloroplast protein profiles are presented. Each lane contains proteins from 4 µg of chlorophyll of chloroplasts. A, lane 1 represents the Bce44B translation product. Import of Bce44B was carried out in the presence of nigericin (lanes 2 and 3), 3.3 mM ATP (lanes 4-6), light with no exogenously added ATP (lanes 7 and 8) and in the dark with no added ATP (lane 9). Samples in lanes 3, 5, and 8 received a post-thermolysin treatment (0.5 µg of protease/µg of chlorophyll). A trypsin treatment (0.75 µg of protease/µg of chlorophyll) was employed in lane 6. B, post-subfractionation of import assays. A crude stromal subfraction is presented in lane 1 and total membranes in lane 2. Sucrose gradient purified outer and inner envelopes subsequent to import are shown in lanes 3 and 4, respectively. All samples in panel B received a post-import thermolysin treatment such as that described in A before subfractionation. C, comparison of in vitro Bce44B translation product (lane 1), imported Bce44B (lane 2), and the immunoreactive 44-kDa bands in total envelopes (lane 3) and total chloroplast samples (lane 4). Lanes 1 and 2 represent a fluorogram of the immunoblot in lanes 3 and 4. The translation product was mixed with the envelope sample in lane 3. The chloroplasts in lane 4 were derived from the import reaction.
Radiolabeled Bce44B precursors were used
directly for assaying import into pea chloroplasts. Thermolysin or
trypsin treatment of reisolated intact chloroplasts was employed to
determine the location of the imported products. Bce44B is targeted to
the chloroplast independent of ATP or light (Fig. 8A, lanes
2-9). The presence of nigericin had no obvious effect on its
targeting. Approximately 50-60% of the added radiolabeled
precursors associated with the plastids, however, most of the
associated precursors were susceptible to a subsequent thermolysin
treatment. Approximately 5% of the associated Bce44B were targeted to a
site that was inaccessible to thermolysin or trypsin (Fig. 7A, lanes 4-6). The low levels of Bce44B
targeted to the envelope in a protease-resistant manner relative to
precursors destined for internal compartments is most likely reflective
of the limited capacity of the envelope membrane for incorporating
additional proteins. Control import experiments conducted with
cytoplasmic proteins such as pyruvate kinase (Wan et al.,
1995) and chloroplast proteins without transit peptides (e.g. Oee1) (Ko and Cashmore, 1989), do not import nor do they bind at
any level to the chloroplast. The affinity Bce44B possesses for
chloroplasts is therefore genuine and did not arise from nonspecific
associations. Bce44B lacks affinity for thylakoid membranes when tested
with isolated thylakoids or when redirected into the chloroplast. ()Recent import studies with deletions of Bce44B confirm the
specificity of its targeting and the existence of the
protease-resistant form. The results of these experiments will be
reported separately.
The suborganellar location of imported protease-resistant Bce44B form was first determined by crude subfractionation (Fig. 8B). Bce44B was found predominantly in the membranous fractions such as crude envelope and thylakoid membranes. The imported protease-resistant Bce44B proteins co-fractionated with both outer and inner envelope membrane fractions (Fig. 8B, lanes 3 and 4), a pattern similar to the one observed for the outer envelope protein Com70 (Wu and Ko, 1993; formerly designated as Sce70). The separation of outer and inner membrane fractions was confirmed by immunoblotting analysis of the same samples with the anti-37-kDa inner membrane protein. The same immunoreactive pattern as that discussed above was observed in these import fractionation experiments (data not shown). Thus like Com70, Bce44B may also be located in the outer envelope membrane. However, the possibility that the same Bce44B may be located in both outer and inner envelopes cannot be ruled out at this point, since unlike the protease sensitivity displayed by imported Com70, a population of Bce44B is protease resistant. Imported Bce44B co-migrated with an authentic 44-kDa B. napus envelope polypeptide and the in vitro translation product, strongly indicating that this protein did not contain a cleavable targeting signal (Fig. 8, lanes 1, 2, and 4).
We have isolated and characterized a full-length cDNA clone
encoding a 44-kDa protein of the B. napus chloroplast envelope
(Bce44B) that possesses a number of unusual characteristics. One
noteworthy feature is that the native 44-kDa envelope proteins appear
as prominent immunostaining bands of total pea envelope protein
profiles, but stains very poorly with Coomassie Blue, suggesting that
these proteins do not possess a high affinity for the dye
molecule. Another interesting characteristic is that Bce44B
is an integral component present in both envelope membranes and yet the
deduced protein sequence does not indicate a typical membranous
polypeptide. The hydropathy plot of the deduced Bce44B protein did not
show any obvious structural characteristics common to membrane
polypeptides such as membrane spanning regions,
instead the
plot indicates that Bce44B is hydrophilic in nature. Interestingly the
same types of features were found in ISP42 and MPI1/ISP45, integral
components of the mitochondrial protein translocation apparatus of the
outer and inner membranes (Vestweber et al., 1989; Baker et al., 1990; Maarse et al., 1992). The amino acid
sequences deduced from the nucleotide sequences available for the two B. napus cDNA inserts (full and partial length) did exhibit a
high degree of similarity to each other indicating that the encoded
proteins are indeed related. Even though they are related at the
protein level, the 3`-untranslated region of the two B. napus cDNA clones are different indicating that there are at least two
distinct genes in the B. napus cultivar used. The distinct
blot hybridization patterns obtained for the genomic DNA and for total
plant cell RNA support the presence of at least two different genes
encoding 44-kDa envelope proteins in B. napus. It is possible
that the two different genes were derived from the two parental lines
used to create the B. napus line employed in our study but the
RNA blot analysis results clearly show that the steady state transcript
levels of the two genes are different and hence they have different
expression profiles. Due to the presence of immunorelated forms of the
44-kDa protein, the relationship between the steady state transcript
level for the two different cDNA clones and the protein levels observed
in the chloroplast envelope is not known at this point and remains to
be determined.
The possibility of multiple related forms of the
44-kDa envelope protein was not limited to the nucleic acid level.
Further analysis of the native 44-kDa proteins in pea envelopes
provided several lines of evidence that at least one immunorelated form
resides in the outer membrane and more abundant immunorelated forms are
present in the inner membrane. The resulting immunoblots of outer and
inner subfractions of pea envelopes indicate that
immunologically-related 44-kDa proteins were present in both membrane
locations. The inner membrane fraction appeared to contain the majority
of the immunorelated 44-kDa proteins observed in the envelope. Further
indication for the dual location of the immunorelated 44-kDa proteins
can be found in the protease treatment results. At least one
immunorelated 44-kDa form is sensitive to thermolysin and gave rise to
a distinct protease-generated 42-kDa band. This 42-kDa
thermolysin-generated product appeared to be protected by the outer
envelope, but was susceptible to trypsin, indicating that at least a
part of an immunorelated 44-kDa protein was accessible to trypsin in
the intermembrane space. The inner membrane immunorelated 44-kDa forms
were resistant to both proteases and were likely protected against
trypsin by the inner envelope. The inner membrane immunorelated 44-kDa
forms were susceptible to thermolysin only when inner membrane
fractions were treated with proteases, subsequent to the purification
of the envelope membranes. Even though the inner immunorelated 44-kDa
forms were accessible to proteases in isolated inner envelopes,
protease treatment gave rise to a distinct proteolytic product. This
suggests that the stromal facing part of the inner proteins was
protected to a large extent by the inner membrane, perhaps by being
embedded in the membrane. Interestingly, trypsin-treated chloroplasts
gave rise to a 30-kDa product that is distinct from the
thermolysin-generated 42-kDa product observed with isolated inner
membranes. Protease digestion in the presence of detergent abolished
all forms of the immunorelated 44-kDa proteins, confirming that the
proteins themselves were completely susceptible in the absence of
membrane association. Immunofluorescence and immunoelectron microscopy
provided two additional lines of evidence that there are multiple
immunorelated 44-kDa forms. The immunofluorescence pattern indicates
that at least one form of the immunorelated 44-kDa envelope protein was
accessible from the outside and was distributed in an uneven fashion in
distinct patches on the surface of the organelle. This interpretation
was reinforced by the immunoelectron microscopy data. The two sets of
microscopy results were consistent with each other as well as with the
immunoblotting and proteolysis experiments. Furthermore, previous
cross-linking and co-immunoprecipitation results indicate that a
translocating chimeric precursor protein can be cross-linked to outer
membrane forms (Com44) as well as to inner membrane forms (Cim44) (Wu et al., 1994). The amount of translocating precursors
cross-linked to outer membrane forms (Com44) versus inner
membrane forms (Cim44) was consistent with the ratios observed in the
subfractionation and immunoblotting experiments. Partial impairment of
Oee1 import by anti-44-kDa IgGs also gave a further indication that at
least one form is potentially accessible from the cytoplasmic side of
the chloroplast and that its limited external location may have a
functional significance. On the whole, the combined data
support the possibility that there are multiple immunorelated forms of
the 44-kDa envelope protein and that at least one immunorelated form is
accessible from the external side of the outer envelope.
The import results of one of these immunorelated 44-kDa proteins, BCE44B, indicate that it is targeted to the chloroplast and the majority of the associated proteins are sensitive to proteases. Only approximately 5-10% of the associated polypeptides were protease-resistant. The lower level of import achieved relative to other well studied chloroplastic precursor proteins most likely reflects the limited capacity of the envelope membrane for accommodating additional new proteins. The similarity of the subfractionation pattern of imported protease-resistant Bce44B to that reported for Com70, an outer membrane protein (Wu and Ko, 1993), suggests that Bce44B may also be an outer envelope protein. However, unlike Com70, the protease-resistant pattern of a population of Bce44B suggests an alternative possibility that the same Bce44B protein is targeted to the organelle and subsequently distributed to both the outer and inner membranes. The dual distribution of the imported Bce44b protein does not appear to be a result of the subfractionation technique since the antibodies against the 37-kDa inner membrane protein immunoreact with a protein band exclusively in the inner membrane fraction. Therefore the Bce44B found in the outer fraction is not due to contamination of the outer membranes with inner membranes. The targeting mechanism of Bce44B is currently the subject of another study and will be reported separately.
The translocation characteristics of Bce44B appear to be distinct from other chloroplast precursors, but are more similar to Omp24, Soe1, Com70, Oep34, and Om14 (Salomon et al., 1992; Li et al., 1992; Wu and Ko, 1993; Fischer et al., 1994; Seedorf et al., 1995). Like these other outer envelope proteins, Bce44B does not appear to contain a cleavable transit sequence. The imported protein co-migrates with the authentic 44-kDa band in Brassica chloroplast envelopes and the in vitro translation product. The bce44B cDNA clone will allow us to further study the translocation pathway of another envelope protein.
Similarly sized polypeptides immunologically-related to Bce44B have
been found in the outer and the inner chloroplast envelopes and have
been implicated to play a role(s) in the translocation of precursor
proteins across the chloroplast envelope membrane (Wu et al.,
1994). These envelope proteins, designated collectively as Com44 (for
the outer membrane forms) and Cim44 (for the inner membrane forms),
were previously demonstrated to be in close physical proximity to
translocation intermediates (Wu et al., 1994). There are
several lines of evidence strongly indicating that Bce44B is a member
of the Com44/Cim44 envelope components of the chloroplast protein
translocation apparatus. First, the antibodies used in the previous
study immunoreact strongly with the Bce44B protein and its
corresponding recombinant phage plaque. Second, the deduced Bce44B
protein sequence is identical to the COOH-terminal polypeptide used to
generate the antibodies employed in the previous chemical cross-linking
study. Third, antibodies generated against the cDNA-encoded Bce44B
protein itself (IgGs made separately against the NH or the
COOH terminus) gave rise to identical chemical
cross-linking/immunoprecipitation results as reported by Wu et
al.(1994). In addition, the developmental profile of the
immunorelated 44-kDa envelope proteins also appears to correlate with
the predicted protein import activity of the various plant tissues. (
)
Due to the multiple characteristics of the 44-kDa chloroplast envelope protein, we are continuing to isolate and characterize more cDNA clones for these proteins from a variety of plants and tissues to gain further insight into these intriguing immunorelated proteins and to determine the relationship between specific proteins and their cDNA clones. The possibility of immunorelated 44-kDa proteins being located in both outer and inner envelope membranes is especially interesting in relation to their potential role in protein import. In the mitochondrial system, the ISP42 component is part of the outer membrane import system and the inner membrane translocation system involves ISP45. However, ISP45 bears no resemblance to ISP42. It appears that in the chloroplast import system immunologically-related proteins may play a role in both the outer and inner import machineries. The potential locale of the 44-kDa immunorelated proteins presented in this study appears to fit into the chloroplast protein import model recently suggested by Schnell and Blobel(1993) featuring two distinct protein conducting channels, one in the outer membrane and one in the inner envelope membrane.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X79091[GenBank].