Soll, Jürgen

Waegemann, Karin

English

Open Access LMU

The Plant Journal (1 992) 2(2), 253-256 SHORT COMMUNICATION A functionally active protein import complex from Jurgen SOH* and Karin Waegemann Botanisches Institut, Christian-Albrechts-Universitat Kiel, OlshausenstraBe 40,0-2300 Kiel 1, Germany Isolated outer chloroplast envelope membranes were solubilized by digitonin and separated on linear sucrose density gradients. A membrane complex was recovered from the gradients and exhibited character- istics of a protein import apparatus, i.e. the interaction of the complex with the precursor polypeptides depends on the presence of a transit sequence, ATP and protease-sensitive components. Furthermore, trans- location intermediates detected in the organellar system are also found after interaction of the precursor polypeptide with the isolated import complex. Introduction A great number of chloroplast proteins are of cytosolic origin and are imported into the organelle in a post- translational event (Keegstra et al., 1989). A challenging question is how precursor proteins are recognized and translocated through the outer and inner chloroplast envelope membranes. The vast majority of precursor proteins seem to follow a common route of translocation, requiring a transit peptide, receptor, and ATP. However, little is known about the membrane components which mediate single steps in the process or their supramolecular arrangement (Cornwall and Keegstra, 1987; Hinz and Flugge, 1988; Kaderbhai etal., 1988). Recent studies have demonstrated that it is possible to isolate a membrane complex from either mitochondria (Kiebler et a/., 1990) or chloroplasts (Waegemann and SOH, 1991) which seems to be involved in protein import. The functional capacity of these membrane complexes was, however, not demon- strated. Here, we report the isolation of a membrane complex from purified outer chloroplast envelopes which is active in recognition and insertion of precursor poly- peptides. Received 9 August 1991; revised 2 October 1991 'For correspondence: (fax +49 431 880 1527). Outer envelope membranes from pea chloroplasts were mildly solubilized by digitonin after incubation with either the precursor form of the small sub-unit of Rubisco (pSSU) or the mature form (SSU) (EC 4.1.1.39). Analysis of the detergent-treated membranes by sucrose density centri- fugation revealed the presence of free pSSU and SSU at the top of the gradient while only pSSU was able to associate with outer envelope membrane components and thus migrated to a higher density in the sucrose gradient (Figure 1). The requirement of a transit sequence for the specific interaction of the outer envelope membrane with a polypeptide destined for the inside of the organelle is clearly demonstrated and extends data obtained earlier (Waegemann and SOH, 1991). To assess the functional capacity of this putative import complex, we solubilized outer envelope membranes with 0.5% digitonin followed by sucrose density centrifugation. Fractions 3-7 from the top of the gradient and 14-19 (0.45 M sucrose), respectively, were pooled and sub- sequently incubated with 35S-labelled pSSU in the presence of Mg-ATP. Partially solubilized and unsolubilized membrane fragments could be recovered from the pellet fraction. The assays were loaded on a second 5 2 0 %  (w/v) sucrose density gradient and spun at 250 000 g for I 5 10 15 20 25 Figure 1. Isolated outer envelope membranes interact with pSSU but not ssu. Purified outer envelope membranes from pea chloroplasts were incubated with 35S-labelled pSSU (m) or SSU (O), respectively (Waegemann and Soll, 1991). Envelope membranes were recovered from the incubation mixture by centrifugation through a sucrose cushion and washed once. Membranes were solubilized and fractionated by a sucrose density gradient (Waegemann and Soll, 1991). Radioactivity was determined by liquid scintillation counting. 253 254 Jurgen So// and Karin Waegemann Figure 2. The translocation complex is present only in fractions 14-19 but not in fractions 3-7 of a typical sucrose density gradient as described in Figure 1. Envelope membranes were solubilized and separated by sucrose density gradient centrifugation. The gradient was fractionated into 25 aliquots. Fraction 3-7 (b) and 14-19 (a) were combined, diluted twofold and in- cubated with 35S-labelled pSSU. The mixture was layered on top of a second 520% (wh) sucrose density gradient and centrifuged overnight. The gradient was fractionated from the top and proteins analysed by SDS- PAGE, silver staining and fluorography. A fluorogram is shown, numbers on top indicate fraction numbers of the second gradients. 14 h. Only the constituents of fractions 14-19 of the first gradient showed the ability to interact with pSSU and shift its mobility to higher density (Figure 2). pSSU did not interact with proteins present in fractions 3-7, and con- sequently the precursor remained in the upper part of the gradient (Figure 2), as did pSSU translation product which was loaded on a mock sucrose density gradient containing buffer instead of solubilized envelope components (data not shown). Post-translational protein uptake into mitochondria and chloroplasts share a number of characteristics common to both systems (Hart1 eta/., 1989; Keegstra et a/., 1989), i.e. a target or transit sequence, proteinaceous receptors exposed on the organellar surface, and hydrolysis of ATP for translocation. The isolated import apparatus from outer envelope membranes exhibits the same character- istics as the organellar system in vitro. Interaction between the putative import complex was detectable only if the precursor form pSSU, but not the mature protein SSU, was used (Figure 3a). These data indicated the presence of a transit sequence-dependent component: the receptor in the complex. Mild protease treatment of outer envelope membrane vesicles prior to digitonin treatment and isola- tion of the complex from the first sucrose density gradient resulted in a pronounced loss of interaction capacity of the complex with pSSU (Figure 3b). Thermolysin-treated complexes showed only a residual binding capacity of 30%, indicating that the untreated complex could indeed contain a receptor for pSSU. Polypeptides which are proteolytically degraded by thermolysin include OEP 86 and OEP 34, both of which are present in the isolated complex one and complex two (Waegemann and SOH, 1991 ; and data not shown). 5 15 25 I00 $ 6 0  2 0  I00 $ 60 2 0  15 17 19 21 2 3  25 27 29 C )  9 12 14 16 18 2 0  2 2  Figure 3. Requirements for an isolated complex from outer chloroplast envelope to interact with pSSU. (a) Interaction of the isolated complex depends on the presence of a transit sequence (m pSSU; 0 SSU). Results were quantified by laser densitometry of the exposed X-ray film. (b) Outer envelope membranes were treated (R) or not treated (B) with 1 pg therrnolysin per 10 pg envelope protein. Membranes were recovered, washed, solubilized and incubated with pSSU as above. Results were quantified as above. (c) pSSU translation product was treated (F2) or not treated (m) with apyrase. The centrifugation time was 4 h instead of overnight. Protein translocation into chloroplasts requires the hydrolysis of ATP (Flugge and Hinz, 1986; Schindler eta/., 1987). To investigate the ATP requirement of pSSU/import complex interaction, the pSSU translation product was depleted of ATP by the action of apyrase (Hwang and Chloroplast import complex 255 Figure 4. pSSU forms a protease-protected complex with envelope poly- peptides. pSSU bound to outer envelope membranes (lanes 1,2), complex one (lanes 3,4) or complex two (lanes 5, 6, 7) was treated in the absence (lanes 1, 2, 5) or presence of thermolysin (lanes 2, 4, 6). Lane 7, as for 6 but with addition of 0.5% Triton X-1 00 and 0.05% SDS. Samples were analysed by SDS-PAGE and fluorography. Schatz, 1989). The removal of ATP resulted in a con- comitant loss of pSSU bound to the complex (Figure 3c). Inclusion of a non-hydrolysable ATP analogue, adenyl- imido-diphosphate (2 mM), in an ATP-depleted pSSU sample did not lower the amount of pSSU in the complex significantly (data not shown). In a recent study (Waegemann and SOH, 1991) trans- location intermediates of pSSU were found using either intact chloroplasts or envelope membranes (see also Figure 4, lanes 1 and 2). Identical protease-protected translocation intermediates pSSU deg 1 and deg 2 could be detected (i) if envelope membranes were loaded with pSSU, solubilized by digitonin, separated by sucrose density gradient centrifugation and treated with protease (Figure 4, lanes 3 and 4), or (ii) if the isolated complex was incubated with pSSU separated on the second sucrose density gradient and treated with protease (Figure 4, lanes 5 and 6). Protease treatment in the presence of 0.5% Triton X-100 and 0.05% SDS resulted in the complete digestion of the protease-protected translocation inter- mediates pSSU deg 1 and deg 2 (Figure 4, lane 7, and data not shown). Furthermore, neither the precursor polypeptide itself nor pSSU obtained from the top of a sucrose gradient (Figure 2a, e.g. fraction 6) are protease resistant per se (data not shown). The results clearly indicate that the protease-protected fragments obtained by thermolysin treatment of the different membrane fractions are true translocation intermediates, providing further evidence that the isolated membrane fraction represents a large part of the translocation machinery of chloroplasts. The protease-protected peptides deg 1 and deg 2 are probably no longer interacting with components at the surface of the complex but it could be that they are trapped in a proteinaceous pore or channel which is formed by the constituents of the import apparatus. The polypeptide pattern of complex two as analysed by SDS-PAGE and silver staining is not altered significantly by the second sucrose density gradient in comparison to complex one (data not shown). Hsc 70 is present in the outer envelope and also in the first and second complex, as determined by immunoblot analysis (Marshall et a/., 1990; Waegemann and SOH, 1991; data not shown). Discussion and conclusions The data presented in this communication demonstrate for the first time that it is possible to isolate a protein trans- location apparatus as a functionally active unit from organelles such as chloroplasts, mitochondria or per- oxisomes. The isolated membrane complex exhibits identical biochemical characteristics to the organellar system, demonstrating that it can be used as a bona fide system to study early events in the import pathway. The import apparatus is made up of a limited number of polypeptides, two of which, OEP 86 and OEP 34, are sensitive to protease, a feature common to a putative receptor. Chloroplastic transit sequences have been postulated to form a random coil and would therefore require guidance by molecular chaperones, e.g. hsc 70, during their passage through the chloroplast envelope membranes (von Heijne and Nishikawa, 1991). Hsc 70, a prominent component in the enriched import apparatus fraction, could act as such a chaperone and coat a protein- aceous pore formed by other constituents. The import pathway of precursor proteins into chloroplasts would therefore be distinct from that of mitochondria in a second important characteristic, which, in contrast to that in chloroplasts, requires a membrane potential (Hart1 et a/., 1989; Hinz and Flugge, 1986; Keegstra et al., 1989; Schindler eta/., 1987). Experimental procedures Solubilization of outer envelope membranes Outer envelope membranes were isolated from pea chloroplasts as previously described (Keegstra and Youssif, 1986; Waegemann and Soll, 1991). Envelope membranes equivalent to 100 pg protein were mildly solubilized in 25 mM Mops-KOH @H 7.2), 1 mM EDTA and 0.5% (w/v) digitonin (Kiebler eta/., 1990) for 10 min at room temperature. The suspension was layered on top of a continuous 5 2 0 %  (w/v) sucrose density gradient and centrifuged for 4 h at 330 000 gin aswinging bucket rotor (Waegemann and Soll, 1991). The gradients were fractionated from the top into 200 pl aliquots. In general, fractions 14-19 were combined and diluted at least twofold with 25 mM Mops-KOH (pH 7.2), 5 mM MgCI2 and, if not otherwise indicated, 500 pM ATP. Labelled precursor protein (1 0 pl) was added and incubated with the gradient fractions for 10 min at room temperature. The mixture was layered on a second 9 ml continuous 5 2 0 %  (w/v) sucrose density gradient (final volume 12 ml) and spun overnight at 250 000 g in a swinging bucket rotor. Gradients were fractionated from the top into 400 pl aliquots. Protein was precipitated by 10% trichloroacetic acid. 256 Jurgen Sol1 and Karin Waegemann Polypeptides were analysed by SDS-PAGE (Laemmli, 1970), silver staining (Ansorge, 1982) and fluorography (Bonner and Laskey, 1974). Incubation of envelope membranes with pSSU Envelope membranes were incubated with pSSU as described by Waegemann and Sol1 (1 991). Membranes were separated from the incubation mixture by centrifugation through a sucrose cushion and washed once prior to thermolysin treatment or solubilization. Protease treatment of envelope membranes either before or after incubation with precursor protein was carried out at 1 pg protease per 10 pg envelope protein at 25°C for 90 sec in the presence of 0.5 mM CaCI2 and 5 mM MgC12. The reaction was stopped by the addition of excess EDTA. Membranes were repurified as above. pSSU containing complex one and complex two were treated with thermolysin at 5 pg ml-’ for 90 sec at 25°C. The reaction was stopped and analysed further as described above. Acknowledgements This work was supported by the Deutsche Forschungsgemein- schaft (SFB 246) and Fonds der Chemischen Industrie. References Ansorge, W. (1982) Fast visualization of protein bands by in- pregnation in potassium permanganate and silver nitrate. Electrophoresis, 82, 235-242. Bonner, W.M. and Laskey, R.A. (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-88. Cornwall, K.L. and Keegstra, K. (1987) Evidence that a chloro- plast surface protein is associated with a specific binding site for the precursor to the small subunit of ribulose-l,5-bisphos- phate carboxylase. Plant Physiol. 85, 780-785. Flugge, U.4. and Hinr, G. (1986) Energy dependence of protein translocation into chloroplasts. Eur. J. Biochem. 160,563-570. Hartl, F.-U., Wanner, N., Nicholson, D.W. and Neupert, W. (1 989) Mitochondria1 protein import. Biochem. Biophys. Acta, 988,1-45. von Heijne, G. and Nishikawa, K. (1991) Chloroplast transit peptides: the perfect random coil? FEBS Lett. 278, 1-3. Hinr, G. and Flugge, U.4. (1988) Phosphorylation of a 51 kDa envelope membrane polypeptide involved in protein transloca- tion into chloroplasts. Eur. J. Biochem. 175, 649-659. Hwang, S.T. and Schatr, G. (1989) Translocation of proteins across the mitochondria1 inner membrane, but not into the outer membrane, requires nucleoside triphosphates in the matrix. Proc. Natl Acad. Sci. USA, 86, 84324436. Kaderbhai, M.A., Pickering, T., Austen, B.M. and Kaderbhai, N. (1 988) A photoactivable synthetic transit peptide labels 30 kDa and 52 kDa polypeptides of the chloroplast inner envelope membrane. FEBS Lett. 232,313-31 6. Keegstra, K., Olsen, L.J. and Theg, S.M. (1 989) Chloroplastic precursors and their transport across the envelope membranes. Ann. Rev. Plant Physiol. 40, 471-501. Keegstra, K. and Youssif, A.E. (1 986) Isolation and characteriza- tion of chloroplast envelope membranes. Methods Enzymol. 118,316-325. Kiebler, M., Waller, R., Sollner, T., Griffiths, G., Hartmann, H., Wanner, N. and Neupert, W. (1990) Identification of a mito- chondrial receptor complex required for recognition and membrane insertion of precursor proteins. Nature, 348, 61 0- 61 6. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 68Ck 685. Marshall, J.S., De Rocher, A., Keegstra, K. and Vierling, E. (1990) Identification of heat shock protein hsp 70 homologues in chloroplasts. Proc. NatlAcad. Sci. USA, 87, 374-378. Schindler, C., Hracky, R. and Soll, J. (1987) Protein transport in chloroplasts: ATP is prerequisite. Zeitschriff Naturforsch. 42c, Waegemann, K. and SOH, J. (1991) Characterization of the protein import apparatus in isolated outer envelopes of chloro- plasts. Plant J. 1, 149-1 58. 103-1 08. 

A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels.

Chloroplast transit peptides: the perfect random coil?

Cleavage of structural proteins during the assembly of the head of bacteriophage T4.

Energy dependence of protein translocation into chloroplasts.

Evidence that a chloroplast surface protein is associated with a specific binding site for the precursor to the small subunit of ribulose-l,5-bisphosphate carboxylase.

Fast visualization of protein bands by inpregnation in potassium permanganate and silver nitrate.

Identification of a mitochondrial receptor complex required for recognition and membrane insertion of precursor proteins.

Identification of heat shock protein hsp 70 homologues in chloroplasts.

Phosphorylation of a 51 kDa envelope membrane polypeptide involved in protein translocation into chloroplasts.

Protein transport in chloroplasts: ATP is prerequisite. Zeitschriff Naturforsch.

Translocation of proteins across the mitochondria1 inner membrane, but not into the outer membrane, requires nucleoside triphosphates in the matrix.

A funcionally active protein complex from chloroplasts

Universität München: Elektronischen Publikationen

The Plant Journal (1 992) 2(2), 253-256 
SHORT COMMUNICATION 
A functionally active protein import complex from 
Jurgen SOH* and Karin Waegemann 
Botanisches Institut, Christian-Albrechts-Universitat Kiel, 
OlshausenstraBe 40,0-2300 Kiel 1, Germany 
Isolated outer chloroplast envelope membranes were 
solubilized by digitonin and separated on linear 
sucrose density gradients. A membrane complex was 
recovered from the gradients and exhibited character- 
istics of a protein import apparatus, i.e. the interaction 
of the complex with the precursor polypeptides depends 
on the presence of a transit sequence, ATP and 
protease-sensitive components. Furthermore, trans- 
location intermediates detected in the organellar 
system are also found after interaction of the precursor 
polypeptide with the isolated import complex. 
Introduction 
A great number of chloroplast proteins are of cytosolic 
origin and are imported into the organelle in a post- 
translational event (Keegstra et al., 1989). A challenging 
question is how precursor proteins are recognized and 
translocated through the outer and inner chloroplast 
envelope membranes. The vast majority of precursor 
proteins seem to follow a common route of translocation, 
requiring a transit peptide, receptor, and ATP. However, 
little is known about the membrane components which 
mediate single steps in the process or their supramolecular 
arrangement (Cornwall and Keegstra, 1987; Hinz and 
Flugge, 1988; Kaderbhai etal., 1988). Recent studies have 
demonstrated that it is possible to isolate a membrane 
complex from either mitochondria (Kiebler et a/., 1990) or 
chloroplasts (Waegemann and SOH, 1991) which seems to 
be involved in protein import. The functional capacity of 
these membrane complexes was, however, not demon- 
strated. Here, we report the isolation of a membrane 
complex from purified outer chloroplast envelopes which 
is active in recognition and insertion of precursor poly- 
peptides. 
Received 9 August 1991; revised 2 October 1991 
'For correspondence: (fax +49 431 880 1527). 
Outer envelope membranes from pea chloroplasts were 
mildly solubilized by digitonin after incubation with either 
the precursor form of the small sub-unit of Rubisco (pSSU) 
or the mature form (SSU) (EC 4.1.1.39). Analysis of the 
detergent-treated membranes by sucrose density centri- 
fugation revealed the presence of free pSSU and SSU at 
the top of the gradient while only pSSU was able to 
associate with outer envelope membrane components 
and thus migrated to a higher density in the sucrose 
gradient (Figure 1). The requirement of a transit sequence 
for the specific interaction of the outer envelope membrane 
with a polypeptide destined for the inside of the organelle 
is clearly demonstrated and extends data obtained earlier 
(Waegemann and SOH, 1991). 
To assess the functional capacity of this putative import 
complex, we solubilized outer envelope membranes with 
0.5% digitonin followed by sucrose density centrifugation. 
Fractions 3-7 from the top of the gradient and 14-19 
(0.45 M sucrose), respectively, were pooled and sub- 
sequently incubated with 35S-labelled pSSU in the 
presence of Mg-ATP. Partially solubilized and unsolubilized 
membrane fragments could be recovered from the pellet 
fraction. The assays were loaded on a second 5 2 0 %  
(w/v) sucrose density gradient and spun at 250 000 g for 
I 5 10 15 20 25 
Figure 1. Isolated outer envelope membranes interact with pSSU but not 
ssu. 
Purified outer envelope membranes from pea chloroplasts were incubated 
with 35S-labelled pSSU (m) or SSU (O), respectively (Waegemann and 
Soll, 1991). Envelope membranes were recovered from the incubation 
mixture by centrifugation through a sucrose cushion and washed once. 
Membranes were solubilized and fractionated by a sucrose density gradient 
(Waegemann and Soll, 1991). Radioactivity was determined by liquid 
scintillation counting. 
253 
254 Jurgen So// and Karin Waegemann 
Figure 2. The translocation complex is present only in fractions 14-19 but 
not in fractions 3-7 of a typical sucrose density gradient as described in 
Figure 1. 
Envelope membranes were solubilized and separated by sucrose density 
gradient centrifugation. The gradient was fractionated into 25 aliquots. 
Fraction 3-7 (b) and 14-19 (a) were combined, diluted twofold and in- 
cubated with 35S-labelled pSSU. The mixture was layered on top of a 
second 520% (wh) sucrose density gradient and centrifuged overnight. 
The gradient was fractionated from the top and proteins analysed by SDS- 
PAGE, silver staining and fluorography. A fluorogram is shown, numbers 
on top indicate fraction numbers of the second gradients. 
14 h. Only the constituents of fractions 14-19 of the first 
gradient showed the ability to interact with pSSU and shift 
its mobility to higher density (Figure 2). pSSU did not 
interact with proteins present in fractions 3-7, and con- 
sequently the precursor remained in the upper part of the 
gradient (Figure 2), as did pSSU translation product which 
was loaded on a mock sucrose density gradient containing 
buffer instead of solubilized envelope components (data 
not shown). 
Post-translational protein uptake into mitochondria and 
chloroplasts share a number of characteristics common 
to both systems (Hart1 eta/., 1989; Keegstra et a/., 1989), 
i.e. a target or transit sequence, proteinaceous receptors 
exposed on the organellar surface, and hydrolysis of ATP 
for translocation. The isolated import apparatus from 
outer envelope membranes exhibits the same character- 
istics as the organellar system in vitro. Interaction between 
the putative import complex was detectable only if the 
precursor form pSSU, but not the mature protein SSU, 
was used (Figure 3a). These data indicated the presence 
of a transit sequence-dependent component: the receptor 
in the complex. Mild protease treatment of outer envelope 
membrane vesicles prior to digitonin treatment and isola- 
tion of the complex from the first sucrose density gradient 
resulted in a pronounced loss of interaction capacity of the 
complex with pSSU (Figure 3b). Thermolysin-treated 
complexes showed only a residual binding capacity of 
30%, indicating that the untreated complex could indeed 
contain a receptor for pSSU. Polypeptides which are 
proteolytically degraded by thermolysin include OEP 86 
and OEP 34, both of which are present in the isolated 
complex one and complex two (Waegemann and SOH, 
1991 ; and data not shown). 
5 15 25 
I00 
$ 6 0  
2 0  
I00 
$ 60 
2 0  
15 17 19 21 2 3  25 27 29 
C )  
9 12 14 16 18 2 0  2 2  
Figure 3. Requirements for an isolated complex from outer chloroplast 
envelope to interact with pSSU. 
(a) Interaction of the isolated complex depends on the presence of a transit 
sequence (m pSSU; 0 SSU). Results were quantified by laser densitometry 
of the exposed X-ray film. 
(b) Outer envelope membranes were treated (R) or not treated (B) with 1 
pg therrnolysin per 10 pg envelope protein. Membranes were recovered, 
washed, solubilized and incubated with pSSU as above. Results were 
quantified as above. 
(c) pSSU translation product was treated (F2) or not treated (m) with 
apyrase. The centrifugation time was 4 h instead of overnight. 
Protein translocation into chloroplasts requires the 
hydrolysis of ATP (Flugge and Hinz, 1986; Schindler eta/., 
1987). To investigate the ATP requirement of pSSU/import 
complex interaction, the pSSU translation product was 
depleted of ATP by the action of apyrase (Hwang and 
Chloroplast import complex 255 
Figure 4. pSSU forms a protease-protected complex with envelope poly- 
peptides. 
pSSU bound to outer envelope membranes (lanes 1,2), complex one (lanes 
3,4) or complex two (lanes 5, 6, 7) was treated in the absence (lanes 1, 2, 
5) or presence of thermolysin (lanes 2, 4, 6). Lane 7, as for 6 but with 
addition of 0.5% Triton X-1 00 and 0.05% SDS. Samples were analysed by 
SDS-PAGE and fluorography. 
Schatz, 1989). The removal of ATP resulted in a con- 
comitant loss of pSSU bound to the complex (Figure 3c). 
Inclusion of a non-hydrolysable ATP analogue, adenyl- 
imido-diphosphate (2 mM), in an ATP-depleted pSSU 
sample did not lower the amount of pSSU in the complex 
significantly (data not shown). 
In a recent study (Waegemann and SOH, 1991) trans- 
location intermediates of pSSU were found using either 
intact chloroplasts or envelope membranes (see also 
Figure 4, lanes 1 and 2). Identical protease-protected 
translocation intermediates pSSU deg 1 and deg 2 could 
be detected (i) if envelope membranes were loaded with 
pSSU, solubilized by digitonin, separated by sucrose 
density gradient centrifugation and treated with protease 
(Figure 4, lanes 3 and 4), or (ii) if the isolated complex was 
incubated with pSSU separated on the second sucrose 
density gradient and treated with protease (Figure 4, lanes 
5 and 6). Protease treatment in the presence of 0.5% 
Triton X-100 and 0.05% SDS resulted in the complete 
digestion of the protease-protected translocation inter- 
mediates pSSU deg 1 and deg 2 (Figure 4, lane 7, and data 
not shown). Furthermore, neither the precursor polypeptide 
itself nor pSSU obtained from the top of a sucrose gradient 
(Figure 2a, e.g. fraction 6) are protease resistant per se 
(data not shown). The results clearly indicate that the 
protease-protected fragments obtained by thermolysin 
treatment of the different membrane fractions are true 
translocation intermediates, providing further evidence 
that the isolated membrane fraction represents a large 
part of the translocation machinery of chloroplasts. The 
protease-protected peptides deg 1 and deg 2 are probably 
no longer interacting with components at the surface of 
the complex but it could be that they are trapped in a 
proteinaceous pore or channel which is formed by the 
constituents of the import apparatus. 
The polypeptide pattern of complex two as analysed by 
SDS-PAGE and silver staining is not altered significantly 
by the second sucrose density gradient in comparison to 
complex one (data not shown). Hsc 70 is present in the 
outer envelope and also in the first and second complex, 
as determined by immunoblot analysis (Marshall et a/., 
1990; Waegemann and SOH, 1991; data not shown). 
Discussion and conclusions 
The data presented in this communication demonstrate 
for the first time that it is possible to isolate a protein trans- 
location apparatus as a functionally active unit from 
organelles such as chloroplasts, mitochondria or per- 
oxisomes. The isolated membrane complex exhibits 
identical biochemical characteristics to the organellar 
system, demonstrating that it can be used as a bona fide 
system to study early events in the import pathway. The 
import apparatus is made up of a limited number of 
polypeptides, two of which, OEP 86 and OEP 34, are 
sensitive to protease, a feature common to a putative 
receptor. Chloroplastic transit sequences have been 
postulated to form a random coil and would therefore 
require guidance by molecular chaperones, e.g. hsc 70, 
during their passage through the chloroplast envelope 
membranes (von Heijne and Nishikawa, 1991). Hsc 70, a 
prominent component in the enriched import apparatus 
fraction, could act as such a chaperone and coat a protein- 
aceous pore formed by other constituents. The import 
pathway of precursor proteins into chloroplasts would 
therefore be distinct from that of mitochondria in a second 
important characteristic, which, in contrast to that in 
chloroplasts, requires a membrane potential (Hart1 et a/., 
1989; Hinz and Flugge, 1986; Keegstra et al., 1989; 
Schindler eta/., 1987). 
Experimental procedures 
Solubilization of outer envelope membranes 
Outer envelope membranes were isolated from pea chloroplasts 
as previously described (Keegstra and Youssif, 1986; Waegemann 
and Soll, 1991). Envelope membranes equivalent to 100 pg protein 
were mildly solubilized in 25 mM Mops-KOH @H 7.2), 1 mM EDTA 
and 0.5% (w/v) digitonin (Kiebler eta/., 1990) for 10 min at room 
temperature. The suspension was layered on top of a continuous 
5 2 0 %  (w/v) sucrose density gradient and centrifuged for 4 h at 
330 000 gin aswinging bucket rotor (Waegemann and Soll, 1991). 
The gradients were fractionated from the top into 200 pl aliquots. 
In general, fractions 14-19 were combined and diluted at least 
twofold with 25 mM Mops-KOH (pH 7.2), 5 mM MgCI2 and, if not 
otherwise indicated, 500 pM ATP. Labelled precursor protein 
(1 0 pl) was added and incubated with the gradient fractions for 10 
min at room temperature. The mixture was layered on a second 
9 ml continuous 5 2 0 %  (w/v) sucrose density gradient (final 
volume 12 ml) and spun overnight at 250 000 g in a swinging 
bucket rotor. Gradients were fractionated from the top into 400 
pl aliquots. Protein was precipitated by 10% trichloroacetic acid. 
256 Jurgen Sol1 and Karin Waegemann 
Polypeptides were analysed by SDS-PAGE (Laemmli, 1970), 
silver staining (Ansorge, 1982) and fluorography (Bonner and 
Laskey, 1974). 
Incubation of envelope membranes with pSSU 
Envelope membranes were incubated with pSSU as described 
by Waegemann and Sol1 (1 991). Membranes were separated from 
the incubation mixture by centrifugation through a sucrose cushion 
and washed once prior to thermolysin treatment or solubilization. 
Protease treatment of envelope membranes either before or after 
incubation with precursor protein was carried out at 1 pg protease 
per 10 pg envelope protein at 25°C for 90 sec in the presence of 
0.5 mM CaCI2 and 5 mM MgC12. The reaction was stopped by the 
addition of excess EDTA. Membranes were repurified as above. 
pSSU containing complex one and complex two were treated 
with thermolysin at 5 pg ml-’ for 90 sec at 25°C. The reaction was 
stopped and analysed further as described above. 
Acknowledgements 
This work was supported by the Deutsche Forschungsgemein- 
schaft (SFB 246) and Fonds der Chemischen Industrie. 
References 
Ansorge, W. (1982) Fast visualization of protein bands by in- 
pregnation in potassium permanganate and silver nitrate. 
Electrophoresis, 82, 235-242. 
Bonner, W.M. and Laskey, R.A. (1974) A film detection method 
for tritium-labelled proteins and nucleic acids in polyacrylamide 
gels. Eur. J. Biochem. 46, 83-88. 
Cornwall, K.L. and Keegstra, K. (1987) Evidence that a chloro- 
plast surface protein is associated with a specific binding site 
for the precursor to the small subunit of ribulose-l,5-bisphos- 
phate carboxylase. Plant Physiol. 85, 780-785. 
Flugge, U.4. and Hinr, G. (1986) Energy dependence of protein 
translocation into chloroplasts. Eur. J. Biochem. 160,563-570. 
Hartl, F.-U., Wanner, N., Nicholson, D.W. and Neupert, W. 
(1 989) Mitochondria1 protein import. Biochem. Biophys. Acta, 
988,1-45. 
von Heijne, G. and Nishikawa, K. (1991) Chloroplast transit 
peptides: the perfect random coil? FEBS Lett. 278, 1-3. 
Hinr, G. and Flugge, U.4. (1988) Phosphorylation of a 51 kDa 
envelope membrane polypeptide involved in protein transloca- 
tion into chloroplasts. Eur. J. Biochem. 175, 649-659. 
Hwang, S.T. and Schatr, G. (1989) Translocation of proteins 
across the mitochondria1 inner membrane, but not into the 
outer membrane, requires nucleoside triphosphates in the 
matrix. Proc. Natl Acad. Sci. USA, 86, 84324436. 
Kaderbhai, M.A., Pickering, T., Austen, B.M. and Kaderbhai, 
N. (1 988) A photoactivable synthetic transit peptide labels 30 
kDa and 52 kDa polypeptides of the chloroplast inner envelope 
membrane. FEBS Lett. 232,313-31 6. 
Keegstra, K., Olsen, L.J. and Theg, S.M. (1 989) Chloroplastic 
precursors and their transport across the envelope membranes. 
Ann. Rev. Plant Physiol. 40, 471-501. 
Keegstra, K. and Youssif, A.E. (1 986) Isolation and characteriza- 
tion of chloroplast envelope membranes. Methods Enzymol. 
118,316-325. 
Kiebler, M., Waller, R., Sollner, T., Griffiths, G., Hartmann, H., 
Wanner, N. and Neupert, W. (1990) Identification of a mito- 
chondrial receptor complex required for recognition and 
membrane insertion of precursor proteins. Nature, 348, 61 0- 
61 6. 
Laemmli, U.K. (1970) Cleavage of structural proteins during the 
assembly of the head of bacteriophage T4. Nature, 227, 68Ck 
685. 
Marshall, J.S., De Rocher, A., Keegstra, K. and Vierling, E. 
(1990) Identification of heat shock protein hsp 70 homologues 
in chloroplasts. Proc. NatlAcad. Sci. USA, 87, 374-378. 
Schindler, C., Hracky, R. and Soll, J. (1987) Protein transport in 
chloroplasts: ATP is prerequisite. Zeitschriff Naturforsch. 42c, 
Waegemann, K. and SOH, J. (1991) Characterization of the 
protein import apparatus in isolated outer envelopes of chloro- 
plasts. Plant J. 1, 149-1 58. 
103-1 08. 


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A funcionally active protein complex from chloroplasts

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