chloroplasts were isolated from the pea leaves of 1012-d - old plants and   purified on silica sol gradients ( waegemann and soll , 1991 ) .
a standard import assay   contained chloroplasts equivalent to 30 g chl in 100 l import mix ( 330   mm sorbitol , 50 mm hepes - koh , ph 7.6 , 3 mm mgso4 , 10 mm methionine , 10 mm cysteine , 20 mm potassium gluconate , 10 mm nahco3 , 2%   ( wt / vol ) bsa , 3 mm atp , and 110% of reticulocyte lysate in vitro  synthesized s - labeled precursor proteins .
chloroplasts were reisolated from the import assay by centrifugation at 4c through a percoll cushion ( 40% [ vol / vol ] percoll in 300 mm   sorbitol , 50 mm hepes - koh , ph 7.6 ) , washed once in hepes - sorbitol   ( waegemann and soll , 1991 ) , and used for further treatments .
prebinding   of precursor proteins to chloroplasts was done under conditions identical   to those described above , except that all steps were carried out at 0c .
chloroplasts were treated with the protease thermolysin in 300 mm   sorbitol , 50 mm hepes - koh , ph 7.6 , 0.5 mm cacl2 for 20 min at 4c at   200 g protease / mg chl .
the reaction was terminated by the addition of 10   mm edta , and chloroplasts were recovered by centrifugation through a   40% percoll cushion and washed once ( joyard et al . , 1983 ; cline et al . ,   1984 ) .
chloroplasts ( equivalent to 200 g chl ) were treated with 200 g   trypsin ( 10700 n-benzoyl - l - arginine ethyl estes
u / mg from bovine pancrease ) in 300 mm sorbitol , 50 mm hepes - koh , ph 8.0 , and 0.1 mm   cacl2 for 1 h at 20c in a final volume of 200 l .
the reaction was stopped   by the addition of 1 mm pmsf and a fivefold excess of soybean trypsin inhibitor ( marshall et al . , 1990 ; lbeck et al . ,
chlorophyll was determined ( arnon , 1949 ) , and   equal amounts of organelles were loaded onto the sds - page on a chl   basis .
after hypotonic lysis in 10 mm hepes - koh , ph 7.6 , chloroplasts were   separated into a soluble stromal and a total membrane fraction by centrifugation for 10 min at 165,000 g. soluble proteins were precipitated with   tca .
import products were analyzed by sds - page ( laemmli , 1970 )   followed by fluorography ( bonner and laskey , 1974 ) .
separation of envelope and thylakoid membranes was achieved after hypotonic lysis of chloroplasts ( see above ) by centrifugation for 30 s at 2,000 g. the resulting pellet contained thylakoid membranes .
the supernatant was once again   centrifuged for 10 min at 165,000 g. soluble proteins were precipitated   with tca and treated as described above .
the pellet fraction , containing   a mixture of envelope and remaining thylakoid membranes , was resuspended in 100 l hepes - koh , ph 7.6 , and layered over a 300-l sucrose   cushion ( 1 m sucrose , 10 mm hepes - koh , ph 7.6 ) .
after centrifugation   for 10 min at 165,000 g , thylakoid membranes had pelleted through the   cushion and were pooled with the above thylakoid fraction .
the supernatant , including the sucrose cushion , was diluted 1:5 with 10 mm hepeskoh , ph 7.6 , and envelope membranes were recovered by centrifugation   for 10 min at 165,000 g. cdnas coding for the different hybrid proteins were constructed by recombinant pcr ( higuchi , 1990 ) .
products were cloned into a vector suitable for in vitro transcription and controlled by dna sequencing ( sanger   et al . , 1977 ) .
the original cdnas coding for piep110 and pssu have   been described ( lbeck et al . , 1996 ; klein and salvucci , 1992 ) . in vitro  
transcription was done using either t7- or sp6-rna polymerase , as outlined before ( salomon et al . , 1990 ) .
proteins were synthesized in reticulocyte lysate in the presence of s - labeled methionine and cysteine ( 1175ci/ mmol ) for 1.5 h at 30c ( salomon et al . ,
overexpression of pssu   was done in escherichia coli bl21 ( de3 ) cells using the pet vector system ( novagen , madison , wi ) ( waegemann and soll , 1995 ) .
chloroplasts were isolated from the pea leaves of 1012-d - old plants and   purified on silica sol gradients ( waegemann and soll , 1991 ) .
a standard import assay   contained chloroplasts equivalent to 30 g chl in 100 l import mix ( 330   mm sorbitol , 50 mm hepes - koh , ph 7.6 , 3 mm mgso4 , 10 mm methionine , 10 mm cysteine , 20 mm potassium gluconate , 10 mm nahco3 , 2%   ( wt / vol ) bsa , 3 mm atp , and 110% of reticulocyte lysate in vitro  synthesized s - labeled precursor proteins .
chloroplasts were reisolated from the import assay by centrifugation at 4c through a percoll cushion ( 40% [ vol / vol ] percoll in 300 mm   sorbitol , 50 mm hepes - koh , ph 7.6 ) , washed once in hepes - sorbitol   ( waegemann and soll , 1991 ) , and used for further treatments .
prebinding   of precursor proteins to chloroplasts was done under conditions identical   to those described above , except that all steps were carried out at 0c .
chloroplasts were treated with the protease thermolysin in 300 mm   sorbitol , 50 mm hepes - koh , ph 7.6 , 0.5 mm cacl2 for 20 min at 4c at   200 g protease / mg chl .
the reaction was terminated by the addition of 10   mm edta , and chloroplasts were recovered by centrifugation through a   40% percoll cushion and washed once ( joyard et al . , 1983 ; cline et al . ,   1984 ) .
chloroplasts ( equivalent to 200 g chl ) were treated with 200 g   trypsin ( 10700 n-benzoyl - l - arginine ethyl estes
u / mg from bovine pancrease ) in 300 mm sorbitol , 50 mm hepes - koh , ph 8.0 , and 0.1 mm   cacl2 for 1 h at 20c in a final volume of 200 l .
the reaction was stopped   by the addition of 1 mm pmsf and a fivefold excess of soybean trypsin inhibitor ( marshall et al . , 1990 ; lbeck et al . ,
chlorophyll was determined ( arnon , 1949 ) , and   equal amounts of organelles were loaded onto the sds - page on a chl   basis .
after hypotonic lysis in 10 mm hepes - koh , ph 7.6 , chloroplasts were   separated into a soluble stromal and a total membrane fraction by centrifugation for 10 min at 165,000 g. soluble proteins were precipitated with   tca .
import products were analyzed by sds - page ( laemmli , 1970 )   followed by fluorography ( bonner and laskey , 1974 ) .
separation of envelope and thylakoid membranes was achieved after hypotonic lysis of chloroplasts ( see above ) by centrifugation for 30 s at 2,000 g. the resulting pellet contained thylakoid membranes .
the supernatant was once again   centrifuged for 10 min at 165,000 g. soluble proteins were precipitated   with tca and treated as described above .
the pellet fraction , containing   a mixture of envelope and remaining thylakoid membranes , was resuspended in 100 l hepes - koh , ph 7.6 , and layered over a 300-l sucrose   cushion ( 1 m sucrose , 10 mm hepes - koh , ph 7.6 ) .
after centrifugation   for 10 min at 165,000 g , thylakoid membranes had pelleted through the   cushion and were pooled with the above thylakoid fraction .
the supernatant , including the sucrose cushion , was diluted 1:5 with 10 mm hepeskoh , ph 7.6 , and envelope membranes were recovered by centrifugation   for 10 min at 165,000 g.
cdnas coding for the different hybrid proteins were constructed by recombinant pcr ( higuchi , 1990 ) .
products were cloned into a vector suitable for in vitro transcription and controlled by dna sequencing ( sanger   et al . , 1977 ) .
the original cdnas coding for piep110 and pssu have   been described ( lbeck et al . , 1996 ; klein and salvucci , 1992 ) . in vitro  
transcription was done using either t7- or sp6-rna polymerase , as outlined before ( salomon et al . ,
proteins were synthesized in reticulocyte lysate in the presence of s - labeled methionine and cysteine ( 1175ci/ mmol ) for 1.5 h at 30c ( salomon et al . , 1990 ) .
overexpression of pssu   was done in escherichia coli bl21 ( de3 ) cells using the pet vector system ( novagen , madison , wi ) ( waegemann and soll , 1995 ) .
the role of iep110 in protein import and its specific topology prompted us to study its import in some detail .
the   protein sequence of iep110 shows the potential to form a   hydrophobic  helix in the nh2 terminus ( amino acids   100140 , fig .
1 b ) , which seems to anchor the protein into   the inner envelope membrane ( lbeck et al . , 1996 ;   kessler et al . , 1996 ) .
initial results had indicated that   piep110 uses the general import pathway into chloroplasts ; however , the translation and import efficiencies of   full - length precursor of iep110 ( piep110 ) were rather   low , so we decided to use a truncated version of piep110 ,   namely the transit peptide of 110 and the nh2-proximal   part of mature iep110 ( tp110 - 110n ; fig .
1 a ) , which comprises amino acids 1269 , to study its import characteristics .   after import of the tp110 - 110n translation product into   intact pea chloroplasts under standard import conditions ,  
the terminally processed form was recovered almost exclusively in the envelope membrane fraction ( fig .
2 a ) . processed 110n was resistant to extraction at ph 11.5 ( 0.1 m   na2co3 ) , indicating a stable insertion into the membrane   similar to in situ ( lbeck et al . , 1996 ) .
no 110n was detectable in the soluble protein fraction of chloroplasts , and   only very little was detectable in the thylakoid membrane   fraction ( fig .
2 a , lane 5 ) . under thylakoid isolation procedures such as those used here ,
these were shown to be   contaminated with envelope membranes ( keegstra and   youssif , 1986 ; joyard et al . , 1991 ) .
therefore , we conclude   that the low amount of 110n found in the thylakoid fraction results from contamination with envelope membranes .   the cooh terminus of iep110 that is exposed to the   intermembrane space in intact pea chloroplasts is inaccessible to the noninvasive protease thermolysin , but is degraded by trypsin , which proteolytically penetrates through   the outer envelope ( lbeck et al . , 1996 ) . imported mature   110n was recovered in a thermolysin - inaccessible but trypsin - accessible manner ( fig . 2 b ) , giving further evidence   that tp110 - 110n is correctly translocated , processed , and   inserted into the inner envelope membrane .
2 c ) using the precursor of the   small subunit of ribulose-1,5-bisphosphate carboxylase   ( pssu ) , a protein localized in the stroma , show that imported mature ssu is inaccessible to trypsin .
these results   indicate that the chloroplastic inner envelope was not penetrated by trypsin under these experimental conditions ,   corroborating earlier results that used the latency of the   hill reaction to measure chloroplast intactness after   trypsin treatment ( lbeck et al . , 1996 ) .
we conclude that   tp110 - 110n can be used as a bona fide substrate to study   the import of piep110 into pea chloroplasts , since both   full - length and truncated precursors are targeted to the inner envelope membrane and exhibit identical behavior to   extraction at ph 11.5 and sensitivity to trypsin .
next , we wanted to know if the targeting information   present in the presequence of iep110 is essential for translocation into the organelle and insertion into the inner envelope .
therefore , the presequences between 110n and   ssu were mutually exchanged , resulting in the constructs   tpssu-110n and tp110-mssu ( see fig .
import experiments into pea chloroplasts clearly show that the transit   peptide of iep110 directs the ssu passenger protein into   the soluble stroma phase , where it is processed and resistant to thermolysin and to trypsin treatment ( fig .
3 a ,   lanes 27 ) . the import efficiency of tp110-mssu , however , is less than 50% of the authentic pssu .
the transit   peptide of ssu also directed the 110n domain of iep110   to chloroplasts in the reverse construct ( fig .
the processed 110n was recovered in a thermolysin - insensitive but trypsin - sensitive manner , indicating   its correct insertion and orientation in the inner envelope   membrane .
we conclude that tp110 contains only stromaltargeting envelope transfer information , and that the topogenic signal for envelope targeting to the inner envelope   membrane and correct insertion is present in the 110n domain of iep110 .  
further evidence for this conclusion results from studies   that demonstrate that tp110 - 110n import is competed for   by the addition of an excess pssu ( fig .
a similar   competition for the import yield can be observed between   the s - labeled pssu translation product and an excess of   e. coli  expressed pssu ( fig . 4 b ) .
e. coli  expressed   pssu was introduced into the import reaction as a ureadenatured protein . while a competition at the level of   translocation was always observed , the binding of reticulocyte lysate  synthesized translation products to the outer   envelope membrane is not competed for to the same extent ( fig .
this might be caused by a certain   amount of nonspecific binding to the chloroplast surface   that can not be competed for by denatured ( i.e. , unfolded )   pssu , which might bypass the primary binding site and   thus compete only at the level of translocation .
4 , a and b ) , however , clearly demonstrate that pssu   and tp110 - 110n use common components of the chloroplast import machinery .  
the stromal processing   protease is a metalloenzyme and can be specifically inhibited by o - phenanthroline ( abad et al . , 1989 ) . in vitro processing of pssu and tp110 - 110n by a stromal extract
demonstrated that both precursor proteins could be converted   to their respective mature forms ( fig .
furthermore , the processing of both preproteins was   inhibited by o - phenanthroline ( fig .
4 , a  c , strongly indicate   that piep110 uses the general import machinery and processing system of the majority of chloroplast - targeted preproteins . as shown above ,
the 110n region contained all the sequence determinants necessary for targeting and insertion   into the inner envelope membrane . to distinguish domains within this region that could be involved in either   targeting or insertion ,
110n was subdivided into n1 , which   contained the most nh2-proximal amino acids of 110n ,   and n2 , which contained the cooh - proximal portion of   the 110n region ( details are outlined in fig .
the sequence of the n2 subdomain was expressed in frame with   tp110 or tpssu , resulting in the hybrid proteins tp110110n2 and tpssu-110n2 .
when import reactions were   performed with either tp110 - 110n2 or tpssu-110n2 , processed 110n2 was recovered exclusively in the soluble   stromal phase ( fig . 5 , a and b , lanes 2 and 3 ) , as was also   indicated by its resistance to either thermolysin or trypsin   treatment ( fig .
the results suggested that the 110n2 domain did not contain sufficient   targeting information for the inner envelope membrane .  
the 110n1 domain , which also comprises the putative   membrane - anchoring  helix , could therefore be solely responsible for membrane targeting .
a hybrid protein was   synthesized which consisted of tp110 fused to the n1 domain , which contained ssu as a cooh - terminal extension and putative reporter for intermembrane space location .
when the tp110 - 110n1-mssu translation product   was imported into chloroplasts , 110n1-mssu was again   largely recovered in the soluble stromal phase .
soluble mature 110n1-mssu was resistant to the proteases thermolysin and trypsin . however ,   membrane - bound 110n1-mssu was resistant to thermolysin but accessible to trypsin , i.e. , 110n1 functions as the   correct topogenic signal , albeit with low efficiency .
the efficiency of membrane targeting and insertion might be   strongly enhanced by the 110n2 domain . to test this notion , tpssu-110n - mssu , which contained both the n1 and n2 subdomains ,
interestingly , processed , mature 110n - mssu was   largely recovered in the membrane fraction ( fig . 5 d ) .  
110n - mssu was resistant to thermolysin but accessible to   trypsin , demonstrating that the hybrid protein attained an   in situ  like membrane orientation ; i.e. , the cooh - proximal portion of 110n - mssu is exposed to the intermembrane space .
between 10 and 20% of processed , mature   110n - mssu was recovered as a soluble protein in a   trypsin - inaccessible location , i.e. , in the stromal phase . up   to this point ,
the data indicate that 110n1 contains the information that anchors the protein into the membrane but   functions with low efficiency , while 110n2 in combination   with 110n1 strongly increases the yield of membraneinserted 110n , maybe because 110n2 contains essential   targeting or sorting information that only works in a cooperative way with 110n1 .
since processed , mature 110n - mssu was detectable in   the membrane as well as in the soluble phase , the following question arose : does 110n - mssu integration into the   inner envelope occur via a soluble intermediate , or is a significant portion of 110n - mssu missorted to the stroma ?  
therefore , s - labeled tpssu-110n - mssu was incubated   with intact chloroplasts in the presence of 3 mm atp for   5 min at 0c to allow binding but no translocation .
organelles were resuspended in 25c tempered   fresh import buffer in the presence of 3 mm atp , and   were incubated further at 25c for various times
6 , a   and b ) . at the earliest time point during the chase period   ( 30 s ) , a significant portion ( 20% ) of the labeled protein   was recovered as membrane - bound precursor , while about   one third was found as soluble processed mature form in   the stroma .
most of the chloroplastbound precursor was imported during the chase period ,   and only 5% of the total labeled protein remained on   the chloroplast surface .
furthermore , the proportion of   soluble 110n - mssu intermediate declined steadily and   was at the brink of detectability after 10 min .
simultaneously , the membrane - recovered portion of 110n - mssu   increased to more than 80% of the initially introduced labeled proteins .
because of the removal of unbound precursor before the beginning of the chase period , we analyzed only the import and insertion of prebound molecules ;   i.e. , the degradation of soluble 110n - mssu can be excluded because , otherwise , we should not be able to detect   an almost constant amount of radioactivity during the entire chase period ( fig .
6 b ) . in the attempts to observe   more of the soluble 110n - mssu protein in the stroma
, the   chase period was initiated by resuspending the chloroplasts in ice - cold import buffer , followed by a slow warming period .
6 , except that at the early time points , 30 s   and 2 min , bound tpssu-110n - mssu made up more than   50% of the total labeled protein , while processed 110nmssu showed a 1:1 distribution between the stroma and   the inner envelope ( data not shown ) .
the final result of   the chase period was identical to that described in fig .
these data clearly demonstrate that a soluble stroma localized intermediate can be chased to a membrane - bound   location with an orientation that most of the protein is exposed to the intermembrane space ( see also fig .
next , we wanted to know if the targeting information   present in the presequence of iep110 is essential for translocation into the organelle and insertion into the inner envelope
. therefore , the presequences between 110n and   ssu were mutually exchanged , resulting in the constructs   tpssu-110n and tp110-mssu ( see fig .
import experiments into pea chloroplasts clearly show that the transit   peptide of iep110 directs the ssu passenger protein into   the soluble stroma phase , where it is processed and resistant to thermolysin and to trypsin treatment ( fig .
3 a ,   lanes 27 ) . the import efficiency of tp110-mssu , however , is less than 50% of the authentic pssu .
the transit   peptide of ssu also directed the 110n domain of iep110   to chloroplasts in the reverse construct ( fig .
the processed 110n was recovered in a thermolysin - insensitive but trypsin - sensitive manner , indicating   its correct insertion and orientation in the inner envelope   membrane .
we conclude that tp110 contains only stromaltargeting envelope transfer information , and that the topogenic signal for envelope targeting to the inner envelope   membrane and correct insertion is present in the 110n domain of iep110 .   further evidence for this conclusion results from studies   that demonstrate that tp110 - 110n import is competed for   by the addition of an excess pssu ( fig .
a similar   competition for the import yield can be observed between   the s - labeled pssu translation product and an excess of   e. coli  expressed pssu ( fig . 4 b ) .
e. coli  expressed   pssu was introduced into the import reaction as a ureadenatured protein . while a competition at the level of   translocation was always observed , the binding of reticulocyte lysate  synthesized translation products to the outer   envelope membrane is not competed for to the same extent ( fig .
this might be caused by a certain   amount of nonspecific binding to the chloroplast surface   that can not be competed for by denatured ( i.e. , unfolded )   pssu , which might bypass the primary binding site and   thus compete only at the level of translocation .
4 , a and b ) , however , clearly demonstrate that pssu   and tp110 - 110n use common components of the chloroplast import machinery .  
the stromal processing   protease is a metalloenzyme and can be specifically inhibited by o - phenanthroline ( abad et al . , 1989 ) .
in vitro processing of pssu and tp110 - 110n by a stromal extract demonstrated that both precursor proteins could be converted   to their respective mature forms ( fig .
furthermore , the processing of both preproteins was   inhibited by o - phenanthroline ( fig .
4 , a  c , strongly indicate   that piep110 uses the general import machinery and processing system of the majority of chloroplast - targeted preproteins .
as shown above , the 110n region contained all the sequence determinants necessary for targeting and insertion   into the inner envelope membrane . to distinguish domains within this region that could be involved in either   targeting or insertion ,
110n was subdivided into n1 , which   contained the most nh2-proximal amino acids of 110n ,   and n2 , which contained the cooh - proximal portion of   the 110n region ( details are outlined in fig .
the sequence of the n2 subdomain was expressed in frame with   tp110 or tpssu , resulting in the hybrid proteins tp110110n2 and tpssu-110n2 . when import reactions were   performed with either tp110 - 110n2 or tpssu-110n2 , processed 110n2
was recovered exclusively in the soluble   stromal phase ( fig . 5 , a and b , lanes 2 and 3 ) , as was also   indicated by its resistance to either thermolysin or trypsin   treatment ( fig . 5 , a and b , lanes 47 ) .
the results suggested that the 110n2 domain did not contain sufficient   targeting information for the inner envelope membrane .  
the 110n1 domain , which also comprises the putative   membrane - anchoring  helix , could therefore be solely responsible for membrane targeting .
a hybrid protein was   synthesized which consisted of tp110 fused to the n1 domain , which contained ssu as a cooh - terminal extension and putative reporter for intermembrane space location .
when the tp110 - 110n1-mssu translation product   was imported into chloroplasts , 110n1-mssu was again   largely recovered in the soluble stromal phase .
however ,   membrane - bound 110n1-mssu was resistant to thermolysin but accessible to trypsin , i.e. , 110n1 functions as the   correct topogenic signal , albeit with low efficiency .
the efficiency of membrane targeting and insertion might be   strongly enhanced by the 110n2 domain . to test this notion , tpssu-110n - mssu , which contained both the n1 and n2 subdomains ,
interestingly , processed , mature 110n - mssu was   largely recovered in the membrane fraction ( fig . 5 d ) .  
110n - mssu was resistant to thermolysin but accessible to   trypsin , demonstrating that the hybrid protein attained an   in situ  like membrane orientation ; i.e. , the cooh - proximal portion of 110n - mssu is exposed to the intermembrane space .
between 10 and 20% of processed , mature   110n - mssu was recovered as a soluble protein in a   trypsin - inaccessible location , i.e. , in the stromal phase . up   to this point , the data indicate that 110n1 contains the information that anchors the protein into the membrane but   functions with low efficiency , while 110n2 in combination   with 110n1 strongly increases the yield of membraneinserted 110n , maybe because 110n2 contains essential   targeting or sorting information that only works in a cooperative way with 110n1 . since processed , mature 110n - mssu was detectable in   the membrane as well as in the soluble phase , the following question arose : does 110n - mssu integration into the   inner envelope occur via a soluble intermediate , or is a significant portion of 110n - mssu missorted to the stroma ?  
therefore , s - labeled tpssu-110n - mssu was incubated   with intact chloroplasts in the presence of 3 mm atp for   5 min at 0c to allow binding but no translocation .
organelles were resuspended in 25c tempered   fresh import buffer in the presence of 3 mm atp , and   were incubated further at 25c for various times
6 , a   and b ) . at the earliest time point during the chase period   ( 30 s ) , a significant portion ( 20% ) of the labeled protein   was recovered as membrane - bound precursor , while about   one third was found as soluble processed mature form in   the stroma .
most of the chloroplastbound precursor was imported during the chase period ,   and only 5% of the total labeled protein remained on   the chloroplast surface .
furthermore , the proportion of   soluble 110n - mssu intermediate declined steadily and   was at the brink of detectability after 10 min .
simultaneously , the membrane - recovered portion of 110n - mssu   increased to more than 80% of the initially introduced labeled proteins . because of the removal of unbound precursor before the beginning of the chase period , we analyzed only the import and insertion of prebound molecules ;   i.e. , the degradation of soluble 110n - mssu can be excluded because , otherwise , we should not be able to detect   an almost constant amount of radioactivity during the entire chase period ( fig .
6 b ) . in the attempts to observe   more of the soluble 110n - mssu protein in the stroma
, the   chase period was initiated by resuspending the chloroplasts in ice - cold import buffer , followed by a slow warming period . in general , the results were similar to those described in fig . 6 , except that at the early time points , 30 s   and 2 min , bound tpssu-110n - mssu made up more than   50% of the total labeled protein , while processed 110nmssu showed a 1:1 distribution between the stroma and   the inner envelope ( data not shown ) .
the final result of   the chase period was identical to that described in fig .
these data clearly demonstrate that a soluble stroma localized intermediate can be chased to a membrane - bound   location with an orientation that most of the protein is exposed to the intermembrane space ( see also fig .
our data demonstrate that chloroplasts have the capability to reexport proteins into the inner envelope after their   complete import into and processing in the stroma .
this is   not only a membrane insertion process , but the translocation machinery involved can export a large polypeptide   chain across the membrane into the intermembrane space .  
the ssu reporter peptide in 110n - mssu has a length of   140 amino acids , which is completely translocated across   the inner envelope into the intermembrane space , as indicated by its complete sensitivity to trypsin .
the components of an export machinery are unknown at the present   time , and it remains to be seen if it shares common constituents with the inner envelope import machinery .
the   identification of a chloroplast - coded , inner envelope localized protein ( sasaki et al . , 1993 ) had indicated previously that an independent transport pathway should exist   for this membrane .
no data exist as to whether this protein   translocates co- or posttranslationally into the inner envelope .
tpssu-110n - mssu is translocated into the inner envelope after processing in the stroma as the mature form ; i.e. ,   the export signal is not present in the transit sequence , but   internal targeting information is necessary .
this topogenic   signal is localized in amino acids 38269 , most likely as a   bipartite signal . the nh2-proximal part ( n1 , amino acids   38149 ) , which also contains the putative membrane anchor region of iep110 , is sufficient for targeting to the inner membrane and proper insertion , albeit with low efficiency .
the n2 region of iep110 ( amino acids 150269 )   can not direct proteins to the inner envelope , but remains   in the stroma upon import .
compartimentation of n1 or   n2 upon import is most likely to be independent of the   stroma - targeting signal used , since both tpssu and tp110   contain only envelope transfer stroma - targeting information . in combination with the n1 region , n2 directs the   protein quantitatively to the inner envelope .
we propose   that n1 contacts the membrane surface very early and that   n2 subsequently initiates the translocation process , e.g. , as   a start transfer signal .
piep110 takes the general chloroplast import pathway ,   as shown by the mutual exchange of transit sequences between piep110 and pssu .
tp110 seems to be less efficient   than tpssu , which supports two- to threefold higher import yields .
simultaneously , more binding to the chloroplast surface is generally detected in constructs that contain tp110 .
import into chloroplasts and integration into the inner envelope of piep110 and tpssu110n are rapid processes , and a soluble intermediate was   beyond detectability .
only when we used tpssu-110nmssu was a soluble intermediate in the translocation   pathway clearly visible . while 50% of mature 110nmssu has already reached the inner envelope after a 30-s   chase period , the results in fig . 5 clearly establish that soluble mature 110n - mssu can be exported into and across   the membrane .
two reasons could explain these differences : ( a ) the passenger protein mssu retards the integration and translocation process because its primary sequence or secondary structure are not well suited for the   export machinery ; and ( b ) the mssu part in 110n - mssu   interacts transiently with its  natural  partners of the   pssu import and assembly pathway , e.g. , chaperonin 60   or the large subunit of ribulose-1,5-bisphosphate carboxylase .
independently of the reason for the kinetic differences in 110n - mssu export , our results show very clearly   that the signal responsible for reexport to the inner envelope protein is present in 110n and is  stronger  than the   potential interaction of mssu with stromal proteins . at   the moment
, it can not be excluded that a fraction of 110nmssu is never released from the membrane before insertion ,   indicating that parallel mechanisms that are used alternatively might exist , or that the currently available experimental approaches are not elaborate enough to resolve   this problem .
lowering the temperature during the critical   chase period did not result in a block of membrane insertion ; only in a general slowdown of the entire process .   therefore , specific inhibitors that block single steps in the   process are needed to describe the sequence of translocation and insertion in detail . however
, our results demonstrate that a reexport machinery exists in the chloroplasts '   stroma that can cooperate faithfully with an inner envelope machinery . in the future , this soluble intermediate   will be used as a tool to search for the components of the   reexport pathway .
the initial protein import experiments   of tpssu-110n - mssu in the presence of nan3 or guanosine nucleotides did not show significant alterations in   the yield of inner envelope 
localized 110n - mssu ( lbeck ,   j. , and j. soll , unpublished data ) .
studies of sorting of proteins to the inner mitochondrial   membrane have suggested that the yeast cytochrome oxidase subunit va uses a 
stop - transfer  mechanism , i.e. ,   one that never leaves the inner membrane , even during   processing by the matrix processing protease ( miller and   cumsky , 1993 ; see also glick et al . , 1992 ) .
sorting of subunit 9 of fo - atpase to the inner membrane of neurospora   mitochondria , however , was proposed to occur after complete translocation into the organelle via a conservative   sorting mechanism ( rojo et al . , 1993 ) .
the soluble matrix   localization of processed mature subunit 9 of fo - atpase   was not analyzed ( rojo et al . , 1993 ; see also stuart and   neupert , 1996 ) .
the soluble intermediate we describe in   this study for the translocation of 110n - mssu into the inner envelope might involve a sorting system that is not   present in mitochondria , e.g. , a sec , nsf , or srp homologue 
( a ) peptide domain combination of various hybrid precursor proteins used in this study and their nomenclature .
( aa ,   amino acids ; tp , transit peptide ; 110n , amino acids 39269 of   piep110 ; 110n1 , amino acids 39149 of iep110 ; 110n2 , amino   acids 150269 of piep110 ) .
inner envelope targeting and insertion information is contained in the   nh2 terminus of iep110 .
a   truncated iep110 precursor ,   namely tp110 - 110n , comprising amino acids 1269 , was   used in standard import experiments with purified pea   chloroplasts ( equivalent to   30 g chl ; a and b ) .
( a )   chloroplasts were separated   into different fractions containing either envelope membranes ( env ) , soluble stroma   ( st ) , and thylakoid membranes ( thy ) .
membranes   were subsequently extracted   with na2co3 at ph 11.5 and   further separated into a pellet ( p ) and soluble fraction   ( s ) .
( b ) inserted , terminally   processed 110n is sensitive   to the protease trypsin but   not to thermolysin .
after completion of a standard import reaction , chloroplasts were either not treated ( lanes 2 and 3 ) or   treated with protease ( thermolysin [ th ] , lanes 4 and 5 ; trypsin   [ try ] , lanes 6 and 7 ) .
organelles were reisolated through a 40%   ( vol / vol ) percoll cushion washed once .
chlorophyll concentration was determined , and equal amounts of organelles were used   for further analysis .
organelles were separated into a soluble   stroma ( st ) and membrane ( m ) fraction .
( c ) pssu is imported   into the chloroplast stroma , where the processed mature form is   resistant to both proteases , thermolysin and trypsin .
all experimental conditions are as decribed in b. s - labeled translation   product ( tl ) is shown as an internal standard , 10% of which was   added to a standard import reaction .
the presequence of   iep110 and ssu were mutually exchanged , and the resulting hybrids , tp110-mssu   and tpssu-110n , were used   under standard chloroplast   import conditions . ( a )
chloroplasts were separated into   a soluble stroma phase and a   total membrane fraction either before or after treatment with thermolysin or trypsin .
( b ) the stroma - directing presequence of ssu ( tpssu ) can not deviate 110n to the stroma .  
import and subfractions of chloroplasts are as decribed in a. all   further experimental conditions and abbreviations are as decribed in fig .
( a ) tp110 - 110n import into pea chloroplasts   was assayed in the absence or   presence of increasing pssu   concentration .
pssuex was   synthesized in e. coli and   added as a urea - denatured   protein to the import assay   ( final urea concentration 80   mm ) .
import experiments   were initiated by the addition   of organelles ( equivalent to   30 g chl ) .
synthesized pssuex competes   the import of reticulocyte lysate made s - labeled pssu   with similar efficiency as   tp110 - 110n .
experimental   conditions are as desribed in   a. ( c ) tp110 - 110n and   pssu are both processed in vitro by a stromal extract .
a stromal processing extract was incubated for either 0 min ( lanes 1 and 4 ) or 90   min ( lanes 2 , 3 and 5 , 6 ) with the respective translation product in the absence ( lane 2 and 5 ) or presence ( lane 3 and 6 ) of 10 mm 1,10   o - phenanthroline
. the n1 and n2   subdomains of iep110 are   both necessary for efficient   envelope targeting and insertion .
( a and b ) amino acids   150269 of iep110 were   fused to the presequence of   either iep110 or ssu , resulting in tp110 - 110n2 and   tpssu-110n2 , respectively .  
s - labeled translation product was incubated with intact   pea chloroplasts ( equivalent   to 30 g chl ) , and the localization of processed , mature   110n2 was tested by chloroplast subfractionation before   ( lanes 2 and 3 ) or after treatment with the proteases thermolysin ( lanes 4 and 5 ) or   trypsin ( lanes 6 and 7 ) .
chloroplasts were subfractionated into a soluble stroma phase ( lanes 2 , 4 , and 6 ) and a membrane fraction ( lanes 3 , 5 , and 7 ) either before ( lanes 2 and 3 ) or after treatment of the organelles with the proteases thermolysin ( lanes 4 and 5 ) or trypsin ( lanes 6 and 7 ) .  
( d ) the tpssu-110n - mssu translation product is imported into the chloroplasts under standard conditions .
110n - mssu can be translocated into the inner envelope membrane via a soluble stromal intermediate .
( a ) s - labeled   tpssu-110n - mssu translation was incubated with intact chloroplasts ( equivalent to 200 g chl in 500 l reaction volume ) at 0c   to allow binding but not import . after 5 min
, the chloroplasts   were reisolated through a 40% percoll cushion , washed , and resuspended in fresh 25c tempered import buffer .
aliquots   ( equivalent to 30 g chl ) were taken at the time intervals indicated , and the translocation reaction was halted by the addition   of ice - cold import buffer .
( b ) quantification of the fluorogram shown in a. different exposures of the x - ray film were   quantified using a laser densitometer .
the sum of radioactivity   found in either the soluble or membrane fraction at 0.5 min was   set to 100% .
 the chloroplastic inner envelope protein of   110 kd ( iep110 ) is part of the protein import machinery in the pea . 
 different hybrid proteins were constructed to assess the import and sorting pathway of   iep110 . 
 the iep110 precursor ( piep110 ) uses the general import pathway into chloroplasts , as shown by the   mutual exchange of presequences with the precursor of   the small subunit of ribulose-1,5-bisphosphate carboxylase ( pssu ) . sorting information to the chloroplastic   inner envelope 
 is contained in an nh2-proximal part of   mature iep110 ( 110n ) . 
 the nh2-terminus serves to anchor the protein into the membrane . 
 large cooh - terminal portions of this protein ( 8090 kd ) are exposed   to the intermembrane space in situ . 
 successful sorting   and integration of iep110 and the derived constructs   into the inner envelope are demonstrated by the inaccessability of processed mature protein to the protease   thermolysin but accessibility to trypsin , i.e. , the imported protein is exposed to the intermembrane space .   a hybrid protein consisting of the transit sequence of   ssu , 
 the nh2-proximal part of mature iep110 , and   mature ssu ( tpssu-110n - mssu ) is completely imported into the chloroplast stroma , from which it can be   recovered as soluble , terminally processed 110nmssu . 
 the soluble 110n - mssu then enters a reexport   pathway , which results not only in the insertion of   110n - mssu into the inner envelope membrane , but   also in the extrusion of large portions of the protein   into the intermembrane space . 
 we conclude that chloroplasts possess a protein reexport machinery for ieps   in which soluble stromal components interact with a   membrane - localized translocation machinery . 
Materials and Methods
Chloroplast Protein Import Assays
Construction of cDNAs Coding for IEP110 Hybrid  Proteins and Synthesis of Labeled Proteins
Results
The IEP110 Transit Peptide Contains Stromal  Targeting Information
Inner Envelope Targeting of IEP110.
Discussion
Figures and Tables