previous articles ( mcbride et al . , 1992 , 1996 ; millar et al . , 1996 ; goping   et al . , 1998 )
these include isolation of rat heart mitochondria , transcription - translation of plasmids encoding vdac and preornithine carbamyl transferase ( poct ) ,   and import of s - labeled translation products into mitochondria in vitro .  
recombinant glutathione s - transferase ( gst)human tom201 - 29 ( formerly named gst-30htom20 ) , lacking the nh2-terminal transmembrane segment of htom20 , was expressed in topp2 cells and purified   ( schleiff et al . , 1997 ) .
protein immobilized on glutathione - sepharose 4b   was washed and suspended in 20 mm napo4 , ph 7.4 , 150 mm nacl , 1 mm   dithiothreitol , 0.5 mm cacl2 , and incubated with 5 g / ml thrombin for 18 h   at 4c , generating htom201 - 29 with an extra gly residue at the nh2 terminus derived from the thrombin cleavage site .
the mixture was passed   through a mono s hr 5/5 column and protein was eluted with a linear   gradient ( buffer a , 10 mm mes , ph 5.0 ; buffer b , 10 mm 3-cyclohexylamino-1-propane sulfonic acid , ph 10 , 500 mm nacl ) .
peak fractions   containing htom201 - 29 were resolved on a superdex 200 column in   10 mm hepes , ph 7.0 , and 100 mm nacl .
fractions containing the purified protein were dialyzed against the same buffer containing 5 mm dtt   and the protein concentrated to 1.5 mg / ml . plasmid encoding gst - htom201 - 29 was manipulated by standard recombinant dna procedures to substitute cys at htom20 codon position   100 with ser and to introduce ser - cys after gly at the thrombin cleavage   site .
the thrombin cleavage product , htom201 - 29/n - gsc / c100s , was   generated and purified as above .
approximately 100 nm large unilamellar vesicles ( luvs ) ( lipid composition : pc , phosphotidylcholine ; pe , phosphotidylethanolamine ; pi , phosphotidylinositol ; ps , phosphotidylserine ; pc / pe / pi / ps at molar ratios of   55:28:13:3 or pc / pe / pi / ps / pe - bmps at molar ratios of 54.5:27.5:13:3:1 )   were prepared in 170 mm sucrose , 20 mm tris acetate , ph 7.0 , and 2 mm   cacl2 ( shahinian and silvius , 1995 ) .
luvs containing -maleimidopropionic acid n - hydroxysuccinimide ester of pe ( pe - bmps ) were incubated   for 12 h at 4c with htom201 - 29/n - gsc / c100s ( 10-fold molar excess   relative to pe - bmps ; coupling efficiency , 8595% ) followed by two 30   min incubations with excess luvs generated in medium lacking sucrose   ( 100 mm nacl , 20 mm tris acetate , ph 7.0 , and 2 mm cacl2 ) to competitively remove unincorporated htom20 .
sucrose - loaded luvs containing   the covalently attached cytosolic domain of htom20 were recovered by   centrifugation at 50,000  g for 60 min in a beckman 75ti rotor .
previous articles ( mcbride et al . , 1992 , 1996 ; millar et al . , 1996 ; goping   et al . , 1998 )
these include isolation of rat heart mitochondria , transcription - translation of plasmids encoding vdac and preornithine carbamyl transferase ( poct ) ,   and import of s - labeled translation products into mitochondria in vitro .  
recombinant glutathione s - transferase ( gst)human tom201 - 29 ( formerly named gst-30htom20 ) , lacking the nh2-terminal transmembrane segment of htom20 , was expressed in topp2 cells and purified   ( schleiff et al . , 1997 ) .
protein immobilized on glutathione - sepharose 4b   was washed and suspended in 20 mm napo4 , ph 7.4 , 150 mm nacl , 1 mm   dithiothreitol , 0.5 mm cacl2 , and incubated with 5 g / ml thrombin for 18 h   at 4c , generating htom201 - 29 with an extra gly residue at the nh2 terminus derived from the thrombin cleavage site .
the mixture was passed   through a mono s hr 5/5 column and protein was eluted with a linear   gradient ( buffer a , 10 mm mes , ph 5.0 ; buffer b , 10 mm 3-cyclohexylamino-1-propane sulfonic acid , ph 10 , 500 mm nacl ) .
peak fractions   containing htom201 - 29 were resolved on a superdex 200 column in   10 mm hepes , ph 7.0 , and 100 mm nacl .
fractions containing the purified protein were dialyzed against the same buffer containing 5 mm dtt   and the protein concentrated to 1.5 mg / ml . plasmid encoding gst - htom201 - 29 was manipulated by standard recombinant dna procedures to substitute cys at htom20 codon position   100 with ser and to introduce ser - cys after gly at the thrombin cleavage   site .
the thrombin cleavage product , htom201 - 29/n - gsc / c100s , was   generated and purified as above .
approximately 100 nm large unilamellar vesicles ( luvs ) ( lipid composition : pc , phosphotidylcholine ; pe , phosphotidylethanolamine ; pi , phosphotidylinositol ; ps , phosphotidylserine ; pc / pe / pi / ps at molar ratios of   55:28:13:3 or pc / pe / pi / ps / pe - bmps at molar ratios of 54.5:27.5:13:3:1 )   were prepared in 170 mm sucrose , 20 mm tris acetate , ph 7.0 , and 2 mm   cacl2 ( shahinian and silvius , 1995 ) .
luvs containing -maleimidopropionic acid n - hydroxysuccinimide ester of pe ( pe - bmps ) were incubated   for 12 h at 4c with htom201 - 29/n - gsc / c100s ( 10-fold molar excess   relative to pe - bmps ; coupling efficiency , 8595% ) followed by two 30   min incubations with excess luvs generated in medium lacking sucrose   ( 100 mm nacl , 20 mm tris acetate , ph 7.0 , and 2 mm cacl2 ) to competitively remove unincorporated htom20 .
sucrose - loaded luvs containing   the covalently attached cytosolic domain of htom20 were recovered by   centrifugation at 50,000  g for 60 min in a beckman 75ti rotor .
standard protein import was conducted by combining rat   heart mitochondria with human vdac synthesized in   reticulocyte lysate . in this system , the preprotein translocation pore can be jammed by partially importing a chimeric protein containing a matrix - targeting signal fused to   dihydrofolate reductase ( dhfr ) .
unfolding of the dhfr   moiety on the cytosolic side of the outer membrane is prevented by the high - affinity dhfr active site inhibitor ,   methotrexate ( mtx ; eilers and schatz , 1986 ; vestweber   et al . , 1989 ) .
in the presence of mtx , excess podhfr   ( sheffield et al . , 1990 ) , a chimeric protein consisting of the   matrix targeting signal of poct fused to dhfr and purified from expressing bacteria , blocked import and processing of poct ( fig .
1 a , lower panel , lanes 3 and 4 ) , whose   processed form otherwise acquires resistance to external   trypsin ( fig .
in addition   to inhibition of poct import , podhfr / mtx inhibited   outer membrane insertion of ytom70(1 - 29)dhfr ( fig .
1   b ) , also designated pomd29 , a chimeric protein comprised of the transmembrane signal - anchor domain of   yeast tom70 fused to dhfr , whose insertion into the   outer membrane of heart mitochondria in vitro has been   documented previously ( li and shore , 1992 ; mcbride et   al . , 1992 ) .
ytom70(1 - 29)dhfr is inserted into the outer   membrane in the nin - ccyto orientation ( li and shore , 1992 ;   mcbride et al . ,
1992 ) and therefore , its import in the absence of competing podhfr was unaffected by mtx   ( not shown ) .  
in contrast to poct and ytom70(1 - 29)dhfr , import   and membrane insertion of vdac in this system , assessed   by the temperature - sensitive acquisition of resistance to   alkaline extraction ( fujiki et al . , 1982 ; goping et al .
1 a , upper panel , compare lanes 7 and 8) , was relatively unaffected by podhfr / mtx ( fig .
1 a , upper   panel , lanes 8 and 9 ) . the slight reduction in vdac import at 5 min effected by podhfr / mtx may relate to   the fact that preproteins like podhfr can stimulate   recruitment of tom20 into the translocation complex ,   thereby competitively reducing the amount of free tom20   in the membrane that is available for interaction with   vdac ( rapaport et al . , 1998 ) .
the failure of partially   translocated podhfr / mtx to block membrane insertion of vdac suggested that vdac may bypass the requirement for the tom40 translocation pore during import . to address this question further , import of vdac
was   examined using isolated yeast mitochondria containing a   temperature - sensitive mutant of tom40 ( kassenbrock et al . ,   1995 ) .
in contrast to control mitochondria , temperature - sensitive tom40 mitochondria were incapable of importing the matrix preprotein form of cytochrome oxidase subunit va ( cox va ) at the nonpermissive temperature   ( 37c ) , as judged by the failure of pcox va to be processed by the mitochondria at 37c ( fig .
1 c , lane 4 ) , while   processing of pcox va was observed at the permissive   temperature of 23c .
in contrast to pcox va , membrane   insertion of vdac occurred at both 23 and 37c ( fig . 1   c ) .
the slight decrease in membrane insertion of vdac   at 37c compared with 23c was similar for both wild - type   and temperature - sensitive tom40 mitochondria ( fig .
1 c ) ,   suggesting that this difference was related to events other   than the temperature - sensitive phenotype of tom40 .
the cytosolic domain of htom20 , htom201 - 29 , when   included as a soluble entity in excess in the import reaction , inhibited import of poct ( fig .
1 a , lower panel ,   compare lanes 8 and 10 ) , presumably because it sequestered the preprotein through direct protein interaction   ( schleiff et al . , 1997 ) and prevented transfer of the preprotein to the translocation machinery .
htom201 - 29 can   also physically interact with vdac ( schleiff et al . , 1997 ) .  
in distinct contrast to poct , however , htom201 - 29 did   not interfere with insertion of vdac into the outer membrane .
1 a ,   upper panel , compare lanes 8 and 10 ) , suggesting that   potential interactions between vdac and htom201 - 29   in the import reaction must be readily reversible .
moreover , the difference in response of poct and vdac to   htom201 - 29 in the import reaction suggested a potential   fundamental difference in the import pathways of the two   proteins .
however , the results also imply that a complex of   vdac and soluble htom201 - 29 can make contact with   the mitochondrial surface in vitro , a suggestion that is   compatible with the observed binding of the cytosolic domain of htom20 to lipid surfaces in vitro ( schleiff and   turnbull , 1998 ; fig .
2 a ) .   to assess the possibility that htom20 can mediate direct   insertion of vdac into a membrane lipid bilayer
, synthetic luvs were created in which the cytosolic domain of   htom20 was linked to the bilayer surface via covalent attachment to pe - pmbs ( fig .
2 a ) . to that end , the protein   was modified to contain a unique cys at the nh2 terminus   of htom201 - 29 ; the other cys located at codon position   100 was converted to ser .
these changes did not influence   the ability of the receptor to interact with either vdac or   poct in vitro ( data not shown ) .
the liposomes had a   phospholipid composition similar to that of total mitochondrial outer membrane in rat liver ( de kroon et al . ,   1997 ) . under the conditions used for mitochondrial protein import reactions with vdac synthesized in reticulocyte lysate
such liposomes were found to efficiently insert   the protein , as determined by acquired resistance to extraction at alkaline ph or with urea ( fig .
moreover , the resulting import product was   resistant to treatment with trypsin ( lane 4 ) , reflective of   vdac in native membranes ( colombini et al . , 1996 ; mannella , 1997 ) .
luvs bearing the htom20 cytosolic domain   did not translocate poct , as judged by the failure of   poct to acquire resistance to trypsin ( fig
. 2 b , lower   panel ) , nor did they insert ytom70(1 - 29)dhfr , as judged   by its extractability from liposomes with 7 m urea ( not   shown ) .
furthermore , vdac that associated with luvs   lacking the htom20 cytosolic domain exhibited facile interliposomal transfer ( fig
as expected , vdac   that was inserted into the luv lipid bilayer by the   htom20 cytosolic domain did not transfer to subsequently   added protein - free luvs .
in contrast , poct that bound   to luvs containing the htom20 cytosolic domain could   subsequently transfer to protein - free luvs , but slower   than poct that bound to luvs lacking htom20 ( t1/2 , 15   versus 1 min ; fig .
this finding is consistent with   the ability of htom20 to physically interact with poct   ( schleiff et al . , 1997 ) .
moreover , the relatively slow dissociation of poct from the htom20 cytosolic domain explains the ability of excess htom201 - 29 to inhibit import   of poct into mitochondria in vitro ( fig .
finally , vdac that had been inserted into luvs by the   htom20 cytosolic domain was examined for its ability to   transport a physiological substrate of this channel protein ,   atp ( rostovtseva and colombini , 1997 ; lee et al . , 1998 ) .  
p]atp before the import reaction , and the subsequent release of atp was measured at   various times after the initiation of the reaction . insertion   of a single functional vdac channel into the vesicle   would be predicted to release encapsulated atp .
release   of radioactive atp commenced immediately upon initiation of vdac insertion into liposomes ( fig .
egress of   atp was dependent on functional vdac , inhibited by a   known antagonist , nadh ( zizi et al .
, 1994 ) , and required   the presence of the htom20 cytosolic domain on the liposome surface to integrate vdac into the lipid bilayer .
in   contrast , the control protein poct did not stimulate atp   export from luvs alone or luvs with htom20 ( fig .
the first indication that a -barrel protein like vdac   might not require a complex preprotein translocation machinery for insertion into a membrane lipid bilayer came   with the observation that purified detergent - solubilized   vdac can spontaneously integrate into planar lipid bilayers as a functional entity .
moreover , in this system insertion   is cooperative as a result of self - mediated stimulation ( xu   and colombini , 1996 ) .
although the lag period for this insertion was relatively long , the results suggest an inherent   ability of the molecule to partition into a lipid bilayer , but   presumably this is because the detergent - extracted entity   is in a favorable state for lipid bilayer integration .
however , in normal physiology , other considerations apply : the   question of membrane specificity ; the problem of competing interactions of vdac with other proteins , specific or   otherwise ; the necessity to maintain a conformation of soluble vdac after release from the ribosome that is compatible with subsequent membrane insertion ; and the requirement for a mechanism to catalyze vdac insertion   upon contact with the mitochondrial outer membrane . in   the import reaction documented here , the appropriate   translocation - competent conformation for vdac may be   supplied by chaperones / factors present in reticulocyte lysate ( smith et al .
the only other minimum requirement was provided by htom20 on the surface of the membrane .
while it is impossible to rule out any involvement   of the tom40 translocation pore , its contribution to   vdac membrane insertion was not detected in the assays   described here , whereas its influence on import of matrix   poct and pcox va , and on membrane insertion of outer   membrane tom70(1 - 29)dhfr was pronounced .
in addition to its role as a receptor in directing newly synthesized   vdac exclusively to the mitochondrial outer membrane   within the context of a whole cell , our results suggest that   htom20 is capable of catalyzing direct insertion of the   protein into the lipid bilayer .
the receptor may accomplish this simply by bringing vdac into close proximity   to the bilayer and/or by triggering release of presumptive   chaperones or other factors from vdac that otherwise   maintain the protein water - soluble . in either scenario ,   vdac may spontaneously insert into the proximate bilayer . alternatively , or in addition , htom20 may play a direct role in guiding the conformational change that allows   the transbilayer -barrel channel to form .
soluble cytosolic domain of htom20 stimulates insertion of vdac into the outer membrane of rat heart mitochondria in vitro by a pathway that is independent of the preprotein   translocation pore .
( a ) s - labeled transcription - translation   products of human vdac and rat poct in reticulocyte lysate   were subjected to standard protein import reactions in the presence of purified rat heart mitochondria for the indicated times at   4c ( lanes 2 and 7 ) or 30c ( lanes 1 , 36 , and 811 ) in the absence   ( lane 1 ) or presence ( lanes 211 ) of mitochondria . also included   in the reactions were 15 g purified podhfr ( sheffield et al . ,  
1990 ) and 2 mm mtx ( lanes 4 , 6 , 9 , and 11 ) , or 15 g purified cytosolic domain of htom20 , htom201 - 29 ( lanes 5 , 6 , 10 , and 11 ) .   at the end of the reactions , the mitochondria were either treated   with trypsin ( poct , lanes 711 ; mcbride et al . , 1992 ) or extracted with 0.1 m naco3 , ph 11.5 ( alkali , vdac lanes 811 ;   goping et al . , 1998 ) .
the bar graph quantifies the radioactive   bands from import reactions of vdac at 30 min , using a power   macintosh 7200/120 and nih image v1.61 image analysis software . shown
the bar numbers refer to the lane numbers in the vdac ( 30 min ) fluorogram .
( b ) import ( 30 min ) of   [ s]vdac , [ s]poct , and [ s]ytom70(1 - 29)dhfr ( previously called pomd29 ; mcbride et al . ,
alkaline - resistant   vdac and ytom70(1 - 29)dhfr and processed poct were analyzed and quantified as in a. ( c ) same as a , except that import   of [ s]vdac or yeast [ s]pcox va was conducted using mitochondria isolated ( daum et al . , 1982 ) from wild - type saccharomyces cerevisiae ( strain d273 - 10b ) or from a yeast strain   ( kky3.3 )
harboring a temperature sensitive mutation in tom40   ( kassenbrock et al . , 1995 ) , and incubated at the nonpermissive   ( 37c ) or permissive ( 23c ) temperatures for tom40 for 60 min   before conducting import reactions .
import was for 30 min at 4 ,   23 , or 37c , as indicated .
p , precursor form of cox va ; m , mature form of cox va .
( a )   schematic illustration of covalent coupling of cytosolic   domain of htom20 ( htom-201 - 29/n - gsc / c100s ; see   materials and methods ) to   the pe - pmbs incorporated   into preformed luvs .
( b )   standard protein import reactions contained sucrose - loaded luvs ( diameter   100 nm ; 0.02 mm lipid ; 1.0   mol% pe - pmbs ) , with or   without covalently attached   htom20 ( 90 nm ) , and were   incubated for 10 min at room   temperature with [ s]vdac   or [ s]poct .
after a 20-fold dilution with reaction   medium , the luvs were recovered by centrifugation at   170,000  g in an airfuge .  
recovery of sucrose - loaded   luvs after centrifugation in   all reactions was > 95% , as   judged by incorporation of   0.1 mol% rhodamine - labeled   pe and detection by fluorescence .
the pellets were subjected to extraction with 0.1 m   naco3 , ph 11.5 ( alkali ;   goping et al .
1998 ) , or with   8 m urea , 40 mm hepes , ph 7.0 ( urea ) .
alternatively , reactions were cooled to 4c and incubated with trypsin ( 1.0 g ) for 20 min , followed by incubation for 20 min with 10 g soybean trypsin inhibitor before dilution and recovery of luvs .
final liposomal pellets were   subjected to 10% sds - page and the products were visualized by fluorography .
( c )   import reactions were conducted with [ s]vdac or [ s]poct in the presence of sucrose - loaded luvs ( 0.07 mm lipid ) with or without attached cytosolic domain of htom20 .
after 10 min , a 10-fold molar excess of plain lipid vesicles lacking sucrose was added for the   indicated time periods , the sucrose - loaded vesicles were isolated by centrifugation as described above , and the associated [ s]vdac or   [ s]poct was quantified by scintillation counting ( expressed as percent of total input ) . shown
sucrose - loaded luvs   with or without covalently attached htom20 and containing   1.8 mm [ p]atp ( 20 ci / mol ) were incubated in a standard protein import reaction ( goping et al . , 1998 ) with vdac or poct   transcription - translation products in the presence or absence of   1 mm nadh .
at the indicated times , 20 vol of import reaction   medium was added , the luvs were collected by centrifugation ,   and radioactivity in the supernatant was determined as in fig .
2   b. shown are the average of the normalized results of three determinations with standard deviations ( maximum radioactivity =   2,100 cpm ) .
the range of total encapsulated atp released from   luvs - tom20 by vdac at 15 min was 510% .
 insertion of newly synthesized proteins into   or across the mitochondrial outer membrane is initiated   by import receptors at the surface of the organelle . 
 typically , this interaction directs the precursor protein into   a preprotein translocation pore , comprised of tom40 .   here 
 , we show that a prominent -barrel channel protein spanning the outer membrane , human voltage- dependent anion - selective channel ( vdac ) , bypasses   the requirement for the tom40 translocation pore during biogenesis . 
 insertion of vdac into the outer membrane is unaffected by plugging the translocation pore   with a partially translocated matrix preprotein , and mitochondria containing a temperature - sensitive mutant   of tom40 insert vdac at the nonpermissive temperature . 
 synthetic liposomes harboring the cytosolic domain of the human import receptor tom20 efficiently   insert newly synthesized vdac , resulting in transbilayer transport of atp .  
 therefore , tom20 transforms   newly synthesized cytosolic vdac into a transmembrane channel that is fully integrated into the lipid bilayer . 
Materials and Methods
General
Recombinant Cytosolic Domain of Human Tom20
Liposomes
Results and Discussion
Figures and Tables