cell culture , transfection , and plasmid constructs  culture 
  and transfection of cos1 cells were performed as previously described 
  ( 2 ) .
mammalian expression 
  plasmids pmcfp - rac1 and pmyfp - rac1 encoding the nh2-terminal fusion 
  of monomeric cyan fluorescence protein ( cfp ) or yellow fluorescence protein 
  ( yfp ) to rac1 as well as rac1 derivatives having the g12v , r66a , c189s , or 6q 
  ( kkrkrk  qqqqqq ) mutations have been 
  described ( 22 , 
  23 ) .
the k186e mutation in 
  rac1 was generated using the stratagene ( la jolla , ca ) quikchange 
  site - directed mutagenesis kit .
plyn - mcfp consists of a 10-amino acid 
  myristoylation / palmitoylation sequence of lyn kinase fused to the 5-end 
  of the mcfp gene ( 6 ) .
rac1(c189s ) or rac1(6q / c189s ) 
  devoid of the geranylgeranylation signal were inserted in frame into the cooh 
  terminus of plyn - mcfp to generate plyn - mcfp - rac1(c189s ) or 
  plyn - mcfp - rac1(6q / c189s ) .
mcfp - gerger and myfp - gerger containing fluorescence 
  protein fused to the caax geranylgeranylation signals , without the 
  upstream polybasic region , were kindly provided by dr .
r. tsien ( university of 
  california , san diego ) 
  ( 24 ) .
plasmids encoding ha - myfp , lyn - ha - myfp , myc - mcfp , and lyn - myc - mcfp fusions 
  were constructed by replacing the enhanced green fluorescent protein gene in 
  pegfp - c1 ( clontech ) with each tag - encoded gene indicated .
various rac1 alleles 
  ( wt , r66a , 6q , c189s , and 6q / c189s ) were then cloned into all four plasmids . 
 
rac1 derivatives used in this study and their 
  properties are described in table 
  1 .
table 1rac1 derivatives used in this studyconstructionspropertiespredicted localization mcfp - rac1 ( wt ) 
   wild type 
   cytoplasm and membrane 
   myfp - rac1 ( wt ) 
   wild type 
   cytoplasm and membrane 
   myfp - rac1 ( 6q ) 
   missing rac1 pbr 
   cytoplasm 
   mcfp - rac1 ( g12v ) 
   gtp hydrolysis - defective 
   cytoplasm and membrane 
   mcfp - rac1 ( r66a ) 
   defective for rhogdi binding 
   predominantly membrane 
   mcfp - rac1 ( c189s ) 
   missing prenylation site 
   nucleus and cytoplasm 
   myfp - rac1 ( k186e ) 
   defective for pip5k binding 
   membrane and cytoplasm 
   mcfp - caax 
   prenylated mcfp 
   membrane 
   myfp - caax 
   prenylated myfp 
   membrane 
   mcfp - rac1 ( g12v ) ( 6q ) 
   gtp hydrolysis - defective ; missing pbr 
   cytoplasm 
   mcfp - rac1 ( g12v ) ( c189s ) 
   gtp hydrolysis defective ; missing prenylation site 
   cytoplasm and nucleus 
   mcfp - rac1 ( g12v ) ( k186e ) 
   gtp hydrolysis - defective ; defective for pip5k binding 
   membrane and cytoplasm 
   lyn - mcfp - rac1 ( 6q ) ( c189s ) 
   myristoylated ; missing pbr and prenylation site 
   membrane 
   lyn - mcfp - rac1 ( c189s ) 
   myristoylated ; missing prenylation site 
   membrane 
   lyn - myfp - rac1 ( c189s ) 
   myristoylated ; missing prenylation site 
   membrane 
   lyn - mcfp - rac1 ( g12v ) ( k186e ) 
   myristoylated ; gtp hydrolysis - defective ; defective for pip5k binding 
   membrane 
   lyn - myfp - rac1 ( g12v ) ( k186e ) 
   myristoylated ; gtp hydrolysis - defective ; defective for pip5k binding 
   membrane 
   lyn - myfp - rac1 ( g12v ) ( c189s ) 
   myristoylated ; gtp hydrolysis - defective ; missing prenylation site 
   membrane 
   lyn - mcfp - rac1 ( g12v ) ( c189s ) 
   myristoylated ; gtp hydrolysis defective ; missing prenylation site 
   membrane 
   lyn - mcfp - rac1 ( 6q ) ( c189s ) 
   myristoylated ; missing pbr and prenylation site 
   membrane 
   lyn - myfp - rac1 ( 6q ) ( c189s ) 
   myristoylated ; missing pbr and prenylation site 
   membrane 
   lyn - mcfp - rac1 ( g12v ) ( 6q ) ( c189s ) 
   myristoylated ; gtp hydrolysis - defective ; missing pbr and prenylation site 
   membrane 
   lyn - myfp - rac1 ( g12v ) ( 6q ) ( c189s ) 
   myristoylated ; gtp hydrolysis - defective ; missing pbr and prenylation site 
   membrane 
   myc - mcfp - rac1 ( wt ) 
   wild type 
   cytoplasm and membrane 
   ha - myfp - rac1 ( wt ) 
   wild type 
   cytoplasm and membrane 
   myc - mcfp - rac1 ( 6q ) 
   missing pbr 
   cytoplasm 
   myc - mcfp - rac1 ( c189s ) 
   gtp hydrolysis - defective ; missing prenylation site 
   cytoplasm and nucleus 
   myc - mcfp - rac1 ( r66a ) 
   defective for rhogdi binding 
   predominantly membrane 
   lyn - myc - mcfp - rac1 ( 6q ) 
   myristoylated ; missing pbr 
   membrane 
   lyn - ha - myfp - rac1 ( 6q ) 
   myristoylated ; missing pbr 
   membrane 
   lyn - ha - myfp - rac1 ( c189s ) 
   myristoylated ; missing prenylation site 
   membrane 
   lyn - myc - mcfp - rac1 ( c189s ) 
   myristoylated ; missing prenylation site 
   membrane 
   lyn - ha - myfp - rac1 ( 6q ) ( c189s ) 
   myristoylated ; missing pbr ; missing prenylation site 
   membrane 
   lyn - myc - mcfp - rac1 ( 6q ) ( c189s ) 
   myristoylated ; missing pbr and prenylation site 
   membrane 
   lyn - myc - mcfp 
   myristoylated mcfp 
   membrane 
 
  lyn - ha - myfp 
 
  myristoylated myfp 
 
  membrane 
   rac1 derivatives used in this study culture of y. pseudotuberculosis infection of mammalian cells and 
  immunofluorescence protection assay of bacterial uptake 
conditions 
  for growth of virulence plasmid - cured y. pseudotuberculosis 
  ypiii(p ) with or without yope or yopt and infection of cos1 have 
  been described ( 22 ) .
the 
  plasmid - cured y. pseudotuberculosis strain lacks the virulence 
  plasmid ( pyv ) that encodes yope and yopt and is efficiently internalized into 
  host cells .
strains that harbor the yope rho family gap 
  ( 22 ) or the yopt family 
  caax protease ( kind gift of dr .
james bliska , suny stony brook ) 
  contain plasmids encoding these proteins as well as the plasmid pyv 
  ( yopt - deficient yope::kan yoph::cam ; 
  referred to as strain yp17 ) to allow translocation of these proteins via the 
  bacterial type iii secretion system 
  ( 22 ) . for bacteria lacking the virulence plasmid , the ypiii(p ) strain 
  was grown logarithmically in luria bertani broth at 26 c until an 
  a600 of 0.7 , prior to inoculation onto cultured mammalian 
  cells ( 22 ) . for strains 
  harboring the pyv plasmid and yop - encoding plasmids ,
bacteria were grown with 
  aeration at 26 c overnight in broth supplemented with 2.5 mm 
  cacl2 and 100 g / ml ampicillin and then subcultured and grown at 
  26 c until a600 of 0.2 . at this point ,
the cultures 
  were shifted to 37 c and aerated for 1 h. a multiplicity of infection of 
  50:1 was used for ypiii(p ) incubations , and a multiplicity of 
  infection of 25:1 was used for other derivatives . for the pyope - expressing 
  plasmid , 0.1 mm isopropyl--d - thiogalactopyranoside 
 
briefly , 
  bacteria appropriately cultured were incubated with transfected cells for 30 
  min at a multiplicity of infection of 50 at 37 c .
after incubation , the 
  adherent cells were analyzed for internalized or surface - bound bacteria as 
  described previously ( 6 ) .
the 
  monolayers were fixed in 3% paraformaldehyde and probed with primary antibody 
  directed against y. pseudotuberculosis , followed by a fluorescent 
  secondary antibody ( anti - igg conjugated to either alexa fluor 594 or cascade 
  blue ) to detect extracellular bacteria .
the cells were then permeabilized 
  ( 2 ) and probed with antibodies 
  directed against the bacteria to allow detection of both intracellular and 
  extracellular bacteria .
fret measurements  the basis of the assay is that association 
  between rac1 derivatives fused to either monomeric cfp or monomeric yfp should 
  be detected as a fret readout .
cos1 cells were first transfected with a 
  combination of various derivatives of mcfp - rac1 and myfp - rac1 and then 
  challenged with bacteria .
the cells were then imaged and analyzed for sensitized fret from cfp to yfp 
  essentially as described ( 22 ) , 
  using correction factors for cfp ( 0.32 ) and yfp ( 0.18 ) for bleed - through from 
  cfp emission and cross - yfp excitation by the fret filter set . to measure fret , 
  images from yfp , cfp , and fret filter sets 
  ( 22 )
sensitized fret was then 
  calculated from these regions , by subtracting the cfp and yfp correction 
  factors , using exactly the same procedure as described previously 
  ( 22 ) .
fret signals were 
  normalized by combination of mcfp and myfp emissions calculated from the cfp 
  and yfp filter sets using the following formula as described 
  ( 25 ) . 
 
( eq.1)\documentclass[10pt]{article } 
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 \begin{document } 
 \begin{equation*}{\mathrm{normalized\;fret}}\hspace{1em}=\hspace{1em}\frac{{\mathrm{sensitized\;\;fret}}}{\sqrt{{\mathrm{cfp}}\hspace{1em}{\times}\hspace{1em}{\mathrm{yfp}}}}\end{equation*}\end{document } 
  determination of myfp - rac1 expression levels relative to endogenous 
  rac1cos1 cells were transfected with myfp - rac1 and cultured 
  overnight .
transfected cells were lifted and subjected to flow cytometry using 
  yfp fluorescence to sort cells into four fractions according to levels of 
  fluorescence ( yfp negative , low , medium , and high ) .
a portion of the sorted 
  cells were plated onto fibronectin - coated coverslips , allowed to adhere for 
  3 h , fixed in 4% paraformaldehyde , and imaged to quantify yfp 
  fluorescence .
the remaining sorted cells were lysed in sample buffer ; 
  lysates were resolved by sds - page , blotted , and probed for rac1 using a 
  monoclonal anti - rac1 antibody ( clone 23a8 ; sigma ) .
immunoprecipitation of myc / ha - tagged mcfp / myfp fusions293 t 
  cells were transfected with 0.5 g of each plasmid in 6-well dishes .
bait 
  constructs were myc - tagged , and prey constructs were ha - tagged .
cells were 
  lysed 24 h post - transfection in 500 l of lysis buffer ( 20 mm 
  tris , ph 7.5 , 150 mm nacl , 1% nonidet p-40 , and complete protease 
  inhibitor mixture ( roche applied science ) ) .
were then incubated at 4 
  c ( with agitation ) for 10 min and spun to remove debris .
50 l of the 
  cleared lysates was saved as the input fraction , and 400 l was applied to 
  washed anti - ha epitope affinity resin ( monoclonal anti - ha ; sigma ) and 
  incubated at 4 c ( with agitation ) for 1 h. the resin was washed three 
  times in wash buffer ( 25 mm tris , ph 7.5 , 30 mm 
  mgcl2 , 40 mm nacl , 0.1% nonidet p-40 ) , and bound 
  proteins were eluted in 100 l of sample buffer .
10 l of eluted 
  proteins ( immunoprecipitate ) as well as 10 l of cleared lysates ( input ) 
  were analyzed by sds - page and western blotting .
input and immunoprecipitated 
  proteins were detected using an anti - myc epitope antibody ( rabbit polyclonal ; 
  santa cruz biotechnology , inc .
, santa cruz , ca ) as well as an anti - ha epitope 
  antibody ( rabbit polyclonal ; santa cruz biotechnology ) .
y . 
  pseudotuberculosis employs a variety of proteins to activate and 
  misregulate rac1 in host cells 
  ( 22 ) . in this report
, we 
  examined whether y. pseudotuberculosis could also control 
  self - association of rac1 ( 21 ) . 
 
previously , self - association was demonstrated in vitro by gel 
  filtration chromatography and immunoprecipitation of differentially tagged 
  rac1 derivatives in an event requiring the cooh - terminal pbr 
  ( fig .
1a ) 
  ( 21 ) . to determine if 
  self - association occurs in cells and can be detected at spatially distinct 
  sites within the cell , rac1 self - association
was analyzed by a fret - based 
  system , using monomeric cfp and yfp derivatives ( mcfp - rac1 and myfp - rac1 ) 
  ( fig .
when 
  coexpressed in the same cells , mcfp - rac1(wt ) and myfp - rac1(wt ) produced fret 
  throughout the cells in all regions except for the nucleus 
  ( fig . 1 , b and 
  c ) .
when normalized to the concentration of rac1 at 
  individual sites in the cell , fret signals ( normalized fret ) were notably 
  stronger at membrane ruffles , indicating enhanced rac1 self - association 
  ( fig .
these elevated fret levels were specific to these sites , 
  because at regions where no ruffles were evident , the lower levels of 
  normalized fret were independent of rac1 concentrations .
gly ( g12 ) , arg ( r66 ) , and 
  lys ( k186 ) are the sites of constitutively active , rhogdi 
  binding - defective , and pip5k binding - defective mutations , respectively . 
 
b , reduced 
  self - association in the absence of membrane localization or loss of the pbr . 
  the mcfp- or myfp - rac1 derivatives noted in b were cotransfected into 
  cos1 cells , and fret measurements were determined ( see  materials and 
  methods  ) .
sensitized fret from 20 regions of interest ( see 
   materials and methods  ) were plotted as a function of the 
  intensity of the mcfp - rac1 donor and myfp - rac1 acceptor .
c - g , 
  left , schematic diagrams for detecting rac1-rac1 self - association 
  using fret .
monomeric forms of cfp and yfp were fused to the nh2 
  terminus of rac1 , as displayed in schematic diagrams .
derivatives in 
  which myfp - rac1 is defective for plasma membrane localization ( e.g. 
  rac1 6q and rac1 c189s ) are defective for interaction with wild - type rac1 on 
  the plasma membrane and thus failed to produce fret .
right , cos1 
  cells were transfected with mcfp - rac1 and myfp - rac1 having the indicated 
  mutations .
the cells were fixed and analyzed by fret using fluorescence 
  microscopy ( see  materials and methods  ) . sensitized
fret 
  ( sens . fret ) corresponds to the total amount of fret , whereas 
  normalized fret ( norm .
fret ) corresponds to the amount of fret per 
  unit of fluorescent protein ( see  materials and methods  ) .
both 
  sensitized fret and normalized fret are displayed as color gradient 
  look - up tables , with the appropriate scale bars below each 
  panel .
white scale bar in 
  g , 10 m ( applies to all panels ) .
gly ( g12 ) , arg ( r66 ) , and 
  lys ( k186 ) are the sites of constitutively active , rhogdi 
  binding - defective , and pip5k binding - defective mutations , respectively . 
 
b , reduced 
  self - association in the absence of membrane localization or loss of the pbr . 
  the mcfp- or myfp - rac1 derivatives noted in b were cotransfected into 
  cos1 cells , and fret measurements were determined ( see  materials and 
  methods  ) .
sensitized fret from 20 regions of interest ( see 
   materials and methods  ) were plotted as a function of the 
  intensity of the mcfp - rac1 donor and myfp - rac1 acceptor .
c - g , 
  left , schematic diagrams for detecting rac1-rac1 self - association 
  using fret .
monomeric forms of cfp and yfp were fused to the nh2 
  terminus of rac1 , as displayed in schematic diagrams .
derivatives in 
  which myfp - rac1 is defective for plasma membrane localization ( e.g. 
  rac1 6q and rac1 c189s ) are defective for interaction with wild - type rac1 on 
  the plasma membrane and thus failed to produce fret .
right , cos1 
  cells were transfected with mcfp - rac1 and myfp - rac1 having the indicated 
  mutations .
the cells were fixed and analyzed by fret using fluorescence 
  microscopy ( see  materials and methods  ) .
fret ) corresponds to the total amount of fret , whereas 
  normalized fret ( norm .
fret ) corresponds to the amount of fret per 
  unit of fluorescent protein ( see  materials and methods  ) .
both 
  sensitized fret and normalized fret are displayed as color gradient 
  look - up tables , with the appropriate scale bars below each 
  panel .
white scale bar in 
  g , 10 m ( applies to all panels ) .
consistent with biochemical data , self - association of rac1 , as detected by 
  fret , required the presence of the pbr on both partners .
co - transfection of 
  mcfp - rac1(wt ) with myfp - rac1(6q ) , which has each of the basic residues in the 
  pbr replaced with gln ( 26 ) , 
  produced a lower fret signal ( fig . 1 , 
  b and d ; 6q ) , confirming the previous 
  published findings that the pbr mediates rac1 self - association in 
  vitro ( 21 ) .
self - association is not due to overexpression of rac1 
  constructs  since any self - association observed by fret could result 
  from aggregation of overexpressed protein , the concentration of rac1 expressed 
  from the transfected plasmids relative to endogenous levels of rac1 was 
  determined .
cos1 cells were transfected with myfp - rac1 and sorted by flow 
  cytometry to determine the concentration of rac1 relative to the amount of yfp 
  fluorescence observed by microscopy .
2a ) , with a peak of untransfected cells and a shoulder of 
  cells having increasing amounts of fluorescence .
the total cell population was 
  collected into four separate fractions consisting of cells with increasing 
  amounts of yfp fluorescence ( neg , lo , med , hi ; 
  fig .
2a ) , and each 
  population was analyzed to determine the amount of myfp - rac1 expression as 
  well as the level of fluorescence using our standard microscopic 
  techniques .
the expression of myfp - rac1 in the yfp - lo fraction was lower than that of 
  endogenous rac1 , based on western blotting with anti - rac1 antibody .
the levels 
  of myfp - rac1 were about 50% of the levels of endogenous rac1 in this 
  population ( fig .
2b ; yfp 
  med and yfp hi ) expressed much higher levels of yfp - rac1 
  compared with endogenous rac1 .
transfectants plated onto coverslips from the 
  yfp - lo population clearly had fluorescence levels similar to those being used 
  for fret analysis ( fig .
when the fluorescence 
  was determined on individual cells from the myfp - lo fraction , the average 
  intensity of individual cells was 500 fluorescence units 
  ( fig .
2d ) , which is 
  about the mean intensity of all the cells used for fret 
  ( fig . 2d ; 
  myfp - rac1(fret ) ) .
in fact , the cell populations that showed 
  overexpression of yfp - rac1 gave fluorescence intensities that were well beyond 
  what was analyzed by fret ( fig . 
 
therefore , a large proportion of the 
  cells analyzed by fret expressed amounts of myfp - rac1 that are at or below 
  endogenous levels of rac1 . we conclude that self - association is not due to 
  overexpression of the fluorescence derivatives .
figure 2.fret analysis of rac1 self - association was performed at physiological 
  rac1 levels .
a , myfp - rac1-transfected cos1 cells were sorted 
  according to fluorescence ( negative ( neg ) , low fluorescence 
  ( lo ) , medium ( med ) , and high ( hi ) ) .
b , 
  sorted cells were lysed and analyzed by western blotting , probing with a 
  monoclonal rac1 antibody ( top ) .
blots were scanned , and the extent of 
  overexpression with respect to endogenous rac1 levels was quantified by 
  densitometry ( bottom ) .
c , sorted cells were plated onto 
  fibronectin - coated coverslips and allowed to adhere .
two 
  representative images are displayed where the med image has been 
  normalized to the fluorescence maximum of the lo image , as indicated 
  by the look - up table .
d , 
  fluorescence intensity of the low and medium fluorescence populations was 
  quantified exactly as the fret analysis .
data are displayed as average yfp 
  fluorescence  s.e . on a per cell basis .
average yfp fluorescence from 
  a typical fret experiment is included in this graph for comparison 
  purposes .
a , myfp - rac1-transfected cos1 cells were sorted 
  according to fluorescence ( negative ( neg ) , low fluorescence 
  ( lo ) , medium ( med ) , and high ( hi ) ) .
b , 
  sorted cells were lysed and analyzed by western blotting , probing with a 
  monoclonal rac1 antibody ( top ) .
blots were scanned , and the extent of 
  overexpression with respect to endogenous rac1 levels was quantified by 
  densitometry ( bottom ) .
c , sorted cells were plated onto 
  fibronectin - coated coverslips and allowed to adhere .
two 
  representative images are displayed where the med image has been 
  normalized to the fluorescence maximum of the lo image , as indicated 
  by the look - up table .
d , 
  fluorescence intensity of the low and medium fluorescence populations was 
  quantified exactly as the fret analysis .
data are displayed as average yfp 
  fluorescence  s.e . on a per cell basis .
average yfp fluorescence from 
  a typical fret experiment is included in this graph for comparison 
  purposes .
myfp - rac1 fusions were expressed in cos1 cells and imaged 
  microscopically for yfp fluorescence .
a , myfp - rac1(wt ) ; b , 
  myfp - rac1(k186e ) , defective for effector ( pip5k ) binding ; c , 
  myfp - rac1(c189s ) , lacking prenylation ; d , lyn - myfp - rac1(c189s ) , 
  myristoylated derivative ; e , myfp - rac1(6q ) , defective pbr ; 
  f , lyn - myfp - rac1(6q)(c189s ) ; g , myfp ; h , lyn - myfp , 
  myristoylated myfp . note that lyn myristoylation targets proteins localized 
  nuclearly ( c ) or perinuclearly ( e ) to the plasma membrane 
  ( f and h , respectively ) .
white scale bar in 
  h , 20 m ( applies to all panels ) .
myfp - rac1 fusions were expressed in cos1 cells and imaged 
  microscopically for yfp fluorescence . a , myfp - rac1(wt ) ; b , 
  myfp - rac1(k186e ) , defective for effector ( pip5k ) binding ; c , 
  myfp - rac1(c189s ) , lacking prenylation ; d , lyn - myfp - rac1(c189s ) , 
  myristoylated derivative ; e , myfp - rac1(6q ) , defective pbr ; 
  f , lyn - myfp - rac1(6q)(c189s ) ; g , myfp ; h , lyn - myfp , 
  myristoylated myfp .
note that lyn myristoylation targets proteins localized 
  nuclearly ( c ) or perinuclearly ( e ) to the plasma membrane 
  ( f and h , respectively ) .
white scale bar in 
  h , 20 m ( applies to all panels ) .
plasma membrane localization of rac1 is not sufficient to promote 
  self - association  the 6q mutation that replaces the pbr has the 
  secondary effect of interfering with plasma membrane localization of rac1 
  ( 14 ) 
  ( fig .
we therefore tested whether blocking plasma membrane 
  localization would affect the ability of rac1 to self - associate .
the c189s 
  mutation , immediately downstream of pbr , prevents geranylgeranylation of rac1 
  at the carboxyl - terminal caax box and resulted in a large pool of the 
  protein localizing in the nucleus , as previously described 
  ( 27 ) 
  ( fig .
this lipid 
  modification is essential for the ras superfamily of small gtpases to anchor 
  onto the plasma membrane ( 28 ) . 
 
based on the fret assay , mcfp - rac1(c189s ) showed no ability to interact with 
  myfp - rac1(wt ) ( fig . 1 , b and 
  e ; c189s ) , indicating the importance of plasma membrane 
  localization for rac1 self - association
. the c189s mutation also blocks rhogdi 
  binding ( 29 ) , raising the 
  possibility that rhogdi could play some role in rac1 self - association . to rule 
  out this possibility ,
the interaction of the rac1(r66a ) mutant was analyzed , 
  which is unable to bind rhogdi but maintains plasma membrane localization 
  ( 22 , 
  30 ) .
mcfp - rac1(r66a ) produced 
  fret with myfp - rac1(wt ) as efficiently as was observed for the interaction 
  between the fluorescent wild type derivatives of rac 
  ( fig . 1 , b and 
  f ; r66a ) .
thus , plasma membrane localization , 
  but not rhogdi binding via the caax geranylgeranylation site , was 
  necessary for rac1 self - association .
figure 4.the polybasic region is not sufficient to allow rac1 self - association of 
  membrane - localized protein .
schematic diagrams on the left 
  represent the pairs of fluorescent proteins used for fret analysis in cos1 
  cells .
derivatives used were mcfp - rac1(wt ) and myfp - rac1(wt ) ( a ) ; the 
  mcfp - gerger and myfp - gerger pair having caax boxes at the carboxyl 
  termini of the fluorescent proteins , lacking all other rac1 sequences 
  ( 24 ) ( b ) ; lyn - mcfp 
  and lyn - myfp , fluorescent proteins localized in the membrane via the 
  nh2-terminal myristoylation site ( c ) ; 
  lyn - mcfp - rac1 - 6q(c189s ) and lyn - myfp - rac1 - 6q(c189s ) , localizing hybrid 
  proteins in the membrane via the nh2-terminal myristoylation site 
  ( d ) ; and lyn - mcfp - rac1(c189s ) and lyn - myfp - rac1(c189s ) , in which 
  caax box defective mutants with intact pbrs are forced into the 
  plasma membrane by the lyn myristoylation signal ( e ) .
constructs 
  encoding these proteins were transfected into cos1 cells , fixed the day after 
  transfection , and subjected to fret microscopy .
representative images of the 
  cfp and yfp channels , along with the sensitized fret ( sens .
fret ) are 
  displayed as color gradient look - up tables , using the displayed 
  scale .
white scale bar in e , 10 m ( applies to 
  all panels ) .
the polybasic region is not sufficient to allow rac1 self - association of 
  membrane - localized protein .
schematic diagrams on the left 
  represent the pairs of fluorescent proteins used for fret analysis in cos1 
  cells .
derivatives used were mcfp - rac1(wt ) and myfp - rac1(wt ) ( a ) ; the 
  mcfp - gerger and myfp - gerger pair having caax boxes at the carboxyl 
  termini of the fluorescent proteins , lacking all other rac1 sequences 
  ( 24 ) ( b ) ; lyn - mcfp 
  and lyn - myfp , fluorescent proteins localized in the membrane via the 
  nh2-terminal myristoylation site ( c ) ; 
  lyn - mcfp - rac1 - 6q(c189s ) and lyn - myfp - rac1 - 6q(c189s ) , localizing hybrid 
  proteins in the membrane via the nh2-terminal myristoylation site 
  ( d ) ; and lyn - mcfp - rac1(c189s ) and lyn - myfp - rac1(c189s ) , in which 
  caax box defective mutants with intact pbrs are forced into the 
  plasma membrane by the lyn myristoylation signal ( e ) . constructs 
  encoding these proteins were transfected into cos1 cells , fixed the day after 
  transfection , and subjected to fret microscopy .
representative images of the 
  cfp and yfp channels , along with the sensitized fret ( sens .
fret ) are 
  displayed as color gradient look - up tables , using the displayed 
  scale .
white scale bar in e , 10 m ( applies to 
  all panels ) . to determine whether plasma membrane localization was sufficient to cause 
  intermolecular fret in this particular assay
, we targeted mcfp / myfp onto the 
  plasma membrane by either an nh2-terminal myristoylation signal 
  from the lyn protein ( 24 ) or 
  the cooh - terminal geranylgeranylation site from rac1 ( see 
  fig .
neither of the two tags could enable the cfp / yfp pair to 
  generate fret ( fig .
5 , a - c and 
  i ) even at high concentrations 
  ( fig . 5 , b and 
  c ) .
this lowered self - association was observed , in 
  addition , with cells that were challenged with y. pseudotuberculosis , 
  since no fret signal could be detected at regions of membrane aggregation 
  resulting from invasin binding or at regions of membrane ruffling ( see below ; 
  fig .
we conclude that plasma membrane localization was not 
  sufficient to cause protein self - association . forcing plasma membrane localization of proteins
that do not interact also 
  was not sufficient to cause protein self - association .
the addition of the lyn 
  myristoylation signal to either rac1(c189s ) 
  ( fig .
rac1(6q / c189s ) ( fig . 
  3 , compare e with f ) resulted in plasma membrane 
  localization of these derivatives .
neither lyn - modified derivative generated a 
  significant fret signal for rac1-rac1 interaction 
  ( fig .
therefore , the aberrant localization of 
  these rac1 derivatives to sites such as the nucleus and perinuclear locales 
  ( fig .
3 ) does not explain the 
  inability of these proteins to self - associate , since even when they are plasma 
  membrane - localized , they could not self - associate .
additionally , gtpase 
  activation does not cause self - association under these conditions , because 
  constitutively active membrane - targeted rac1 variants lacking the appropriate 
  carboxyl - terminal sequences fail to generate a fret signal 
  ( fig .
5 , f , g , and 
  i ) . lowering affinity for effectors reduces rac1 
  self - association  the pbr has been characterized to have a role in 
  binding effectors , such as phosphatidylinositol-4-phosphate 5-kinase- 
  ( pip5k ) and protein kinase c - related kinase 1 
  ( 7 , 
  31 ) . to determine whether 
  disruption of effector recognition reduces rac1 self - association
, we 
  introduced the k186e mutation into rac1 , which has been demonstrated to block 
  the binding of pip5k without affecting plasma membrane localization of 
  rac1 ( 7 ) 
  ( fig .
3b ) . a lower 
  level of mcfp - rac1(k186e ) self - association was observed relative to wild type 
  ( figs .
1 g and 
  5 , h and i ) . 
  therefore , maximum rac1 self - association depended on sequence determinants 
  that are also important for effector binding ( residue lys ) as 
  well as geranylgeranylation ( residue cys ) .
formation of a stable rac1-rac1 complex requires the presence of the 
  pbr and prenylation signal  to support the data from the fret assay , 
  an independent test of rac1 self - association was performed using a 
  coimmunoprecipitation procedure ( fig . 
 
the power of the fret readout is that it provides 
  spatial information and may allow the detection of transient interactions .
if 
  the observed self - association is transient , then it may not survive other 
  detection strategies , such as immunoaffinity techniques , that require slow 
  dissociation rates . to determine
if interaction could be observed using more 
  restrictive conditions , several of the constructs used in the microscopic 
  assay were tagged with either myc or ha epitopes and expressed in mammalian 
  cells ( fig .
two constructs were co - transfected into 293 t cells , each 
  having different tags to allow detection of self - association .
extracts from 
  the transfectants were then subjected to immunoprecipitation with anti - ha , 
  followed by immunoblotting with anti - myc , to determine if a fraction of the 
  myc - tagged partner was found in the precipitate ( see  materials and 
  methods  ) . when ha - tagged cfp - rac1(wt ) was immunoprecipitated , it was 
  able to pellet either myc - tagged yfp - rac1(wt ) or myc - tagged yfp - rac1(r66a ) 
  based on immunoblotting , consistent with the fret observations 
  ( fig .
on the 
  other hand , any disruption of the prenylation site ( rac1(c189s ) ) or the pbr 
  ( rac1(6q ) ) was sufficient to interfere with co - immunoprecipitation 
  ( fig .
this 
  loss of association was true even if only one of the partners was lacking the 
  critical carboxyl - terminal sequences ( fig . 
  6 , b and f ; wt + 6q and 
  wt + c189s ) or if membrane localization was forced by adding 
  a lyn myristoylation site ( fig . 6 , 
  b and f ; lyn constructs ) .
therefore , there is 
  concordance between the two assays for detecting interaction , since rac1 
  self - association is sufficiently long lived to survive detergent extraction 
  and immunoprecipitation .
furthermore , these results provide direct evidence 
  that rac1 self - association requires the pbr and cooh - terminal prenylation 
  signal . figure 5.rac1 self - association of rac1(wt ) is independent of expression 
  levels .
a - h , cos1 cells were cotransfected with 
  mcfp - rac1 and myfp - rac1 constructs , and rac1 self - association was determined 
  by fret in two regions of interest ( roi ) within each cell .
each data 
  point represents results from a single cell , and data are plotted for 20 cells 
  per construct ( see  materials and methods  ) .
sensitized fret 
  determinations were then normalized to the total concentration of both 
  derivatives , calculated as ( mcfp - rac1  myfp - rac1 ) , and 
  data were plotted as a function of the concentration of the derivatives in 
  each of the regions of interest .
data are plotted 
  as the mean of the normalized fret determinations  s.e . ; pooling data 
  from all of the analyzed transfectants are displayed in a - h .
a - h , cos1 cells were cotransfected with 
  mcfp - rac1 and myfp - rac1 constructs , and rac1 self - association was determined 
  by fret in two regions of interest ( roi ) within each cell .
each data 
  point represents results from a single cell , and data are plotted for 20 cells 
  per construct ( see  materials and methods  ) .
sensitized fret 
  determinations were then normalized to the total concentration of both 
  derivatives , calculated as ( mcfp - rac1  myfp - rac1 ) , and 
  data were plotted as a function of the concentration of the derivatives in 
  each of the regions of interest .
data are plotted 
  as the mean of the normalized fret determinations  s.e . ;
pooling data 
  from all of the analyzed transfectants are displayed in a - h .
invasin binding results in localized rac1 
  self - association  engagement of 1 integrin receptors by the 
  y. pseudotuberculosis outer membrane protein invasin triggers the 
  recruitment of rac1 onto the phagosomal membrane 
  ( 6 ) . to examine the degree of 
  self - association of rac1 in response to invasin binding
, we visualized rac1 
  clustered about y. pseudotuberculosis associated with cos1 after a 
  30-min incubation with bacteria .
to analyze self - association , cells were 
  challenged with a bacterial strain that expressed invasin as the only adhesin 
  and lacks the ability to translocate rac1-inactivating yops 
  ( 33 ) .
fret analysis showed 
  that the mcfp - rac1 recruited around nascent phagosomes had elevated levels of 
  self - association in comparison with the nearby rac1 located at regions distal 
  from the phagosomes ( fig . 
 
rac1 on membrane ruffles displayed similar higher 
  efficiency of self - association ( data not shown ; 
  fig .
this enhanced 
  self - association was not due to simple recruitment of mcfp - rac1 and myfp - rac1 
  to the active sites , because the fret levels were still elevated after the 
  fret signals were normalized against the concentration of mcfp - rac1 , 
  indicating that the fraction of rac1 undergoing self - interaction is higher at 
  the site of bacterial adhesion than at other sites 
  ( fig .
in addition , the increased fret signal was 
  dependent on the presence of rac1 in the fusion constructs as well as the 
  caax motif .
when the simple mcfp - gerger / myfp - gerger pair or the 
  lyn - mcfp - rac1(c189s)/lyn - myfp - rac1(c189s ) pair was analyzed , neither was able 
  to generate a fret signal at any site in the host cells even on nascent 
  phagosomes associated with sites of bacterial adhesion 
  ( fig .
this is despite the fact that the 
  caax - containing constructs showed evidence of concentration about the 
  phagosomal cups ( fig .
therefore , enhanced rac1 self - association was not a 
  result of increased membrane density resulting from invasin engagement of 
  integrins but was a property of the presence of an intact rac1 protein .
figure 6.stable self - association of rac1 derivatives shows identical profile to 
  fret assay .
a , ha ( yfp derivatives ) or myc ( cfp derivatives ) 
  epitope tags were introduced into each of the constructs used in the fret 
  assays and used in co - immunoprecipitation experiments to detect stable 
  self - association . displayed
to 
  measure self - association , the noted cfp and yfp derivatives were 
  co - transfected into 293 t cells , and lysates were subjected to 
  immunoprecipitation ( ip ) with anti - ha igg to precipitate yfp 
  derivatives .
association with the noted cfp derivatives was detected after gel 
  fractionation and western blotting ( ib ) with anti - myc igg 
  ( b ) , and the total amount precipitated was determined by blotting 
  with ha ( d ) . as controls to determine relative efficiency of 
  immunoprecipitation , the lysates were probed with anti - myc ( c ) or 
  anti - ha ( e ) , loading 25% of the total fraction available for 
  immunoprecipitation .
f , quantitation of the 
  fraction of rac1 that self - associates . bands from b and c 
  were subjected to densitometry scanning to determine the fraction of 
  myc - tagged protein that associated with ha - tagged rac1 .
a , ha ( yfp derivatives ) or myc ( cfp derivatives ) 
  epitope tags were introduced into each of the constructs used in the fret 
  assays and used in co - immunoprecipitation experiments to detect stable 
  self - association . displayed
to 
  measure self - association , the noted cfp and yfp derivatives were 
  co - transfected into 293 t cells , and lysates were subjected to 
  immunoprecipitation ( ip ) with anti - ha igg to precipitate yfp 
  derivatives .
association with the noted cfp derivatives was detected after gel 
  fractionation and western blotting ( ib ) with anti - myc igg 
  ( b ) , and the total amount precipitated was determined by blotting 
  with ha ( d ) . as controls to determine relative efficiency of 
  immunoprecipitation , the lysates were probed with anti - myc ( c ) or 
  anti - ha ( e ) , loading 25% of the total fraction available for 
  immunoprecipitation .
f , quantitation of the 
  fraction of rac1 that self - associates . bands from b and c 
  were subjected to densitometry scanning to determine the fraction of 
  myc - tagged protein that associated with ha - tagged rac1 .
removal of the caax motif by yersinia yopt protease destroys rac1 
  self - association  the above data argue that the caax 
  prenylation motif and the pbr collaborate to promote rac1 self - association . 
 
one possibility for how this occurs is that membrane insertion resulting from 
  prenylation facilitates close contact between rac1 monomers , stimulating pbr 
  self - association .
the other possibility is that insertion in the membrane is 
  required to maintain self - association . to determine if membrane insertion was 
  required for maintenance of the interaction
, we analyzed the consequences of 
  translocating the y. pseudotuberculosis yopt protein into host cells 
  on rac1 self - association .
the yopt protease activity targets 
  membrane - localized rac1 ( 13 ) , 
  cleaving just upstream of the caax box , releasing rac1 from the 
  membrane .
if the role of the prenyl group is limited to initiating rac1 
  association , then protein released from the membrane should continue to 
  self - associate . to test this model ,
transfectants expressing the 
  mcfp - rac1/myfp - rac1 pair were challenged with a yopt - expressing y. 
  pseudotuberculosis strain , and the amount of fret between the rac1 pairs 
  was determined ( 22 ) . within 1 
  h of incubation with y. pseudotuberculosis
, yopt had reduced rac1 
  self - association , since no fret signal could be detected between the rac1 
  pairs ( fig .
7d ) . 
  therefore , because rac1 rapidly lost self - association after release from the 
  membrane , localization of the protein in the membrane via the prenyl groups 
  was required to maintain this relationship .
furthermore , the loss of 
  self - association after release from the membrane could not be due to 
  competition for binding by rhogdi , because removal of the prenyl group 
  prevents recognition by rhogdi 
  ( 32 ) .
loss of self - association is not the cause of defective signaling that 
  results from alterations in the polybasic region  to investigate the 
  function of the rac1 cooh terminus in invasin - promoted uptake , a strategy was 
  pursued in which mutated plasmid - encoded rac1 genes were introduced into cell 
  lines that expressed endogenous rac1 . to this end
yope is translocated into 
  mammalian cells and subsequently inactivates endogenous rac1 , thus inhibiting 
  the phagocytosis of the bacterium .
this uptake inhibition can be overcome by 
  the exogenous expression of a gap - insensitive ( constitutively active ) rac1 
  allele .
a number of rac1 pbr mutants were introduced onto the constitutively 
  active rac1v12 allele and were transfected into cos1 cells .
cells were 
  challenged with yope - expressing y. pseudotuberculosis ( strain 
  yp17/pyope ) , and the extent of bacterial uptake was quantified .
cos1 cells were transfected with mcfp - rac1 and myfp - rac1 constructs 
  ( a and d ) , mcfp - gerger and myfp - gerger ( b ) , or 
  lyn - mcfp - rac1(c189s ) and lyn - myfp - rac1(c189s ) ( c ) . transfected cells 
 
were challenged the next day with a 30-min incubation of ypiii(p ) 
  ( a - c ) or yp17/pyopt ( d ) , followed by immunostaining of 
  extracellular bacteria ( ext .
bacteria ) bound onto host 
  cells . sensitized and normalized fret readings ( sens .
fret , respectively ) were determined as described 
  ( see  materials and methods  ) .
e , mean normalized fret levels at nascent phagosomes were 
  plotted comparing mcfp - rac1/myfp - rac1 and mcfp - gerger / myfp - gerger as described 
  in the legend to fig . 
 
white scale bar in d , 5 m 
  ( applies to all panels ) .
cos1 cells were transfected with mcfp - rac1 and myfp - rac1 constructs 
  ( a and d ) , mcfp - gerger and myfp - gerger ( b ) , or 
  lyn - mcfp - rac1(c189s ) and lyn - myfp - rac1(c189s ) ( c ) . transfected cells 
 
were challenged the next day with a 30-min incubation of ypiii(p ) 
  ( a - c ) or yp17/pyopt ( d ) , followed by immunostaining of 
  extracellular bacteria ( ext .
bacteria ) bound onto host 
  cells . sensitized and normalized fret readings ( sens .
fret , respectively ) were determined as described 
  ( see  materials and methods  ) .
e , mean normalized fret levels at nascent phagosomes were 
  plotted comparing mcfp - rac1/myfp - rac1 and mcfp - gerger / myfp - gerger as described 
  in the legend to fig . 
 
white scale bar in d , 5 m 
  ( applies to all panels ) .
the 
  indicated plasmids expressing various rac1v12 mutants were used to transfect 
  cos1 cells . all constructs except mcfp - rac1(v12 ) were defective in 
  self - association ( figs . 3 and 
  4 ) , whereas only 
  mcfp - rac1(v12)(c189s ) and mcfp - rac1(v12)(6q ) were defective in localizing on 
  the plasma membrane .
24 h after transfection , cells were challenged with 
  yp17/pyope for 30 min , fixed , and then immunostained for extracellular 
  bacteria and internalized bacteria .
uptake efficiency of 50 cfp - positive cells 
  from each of the three coverslips was determined and summarized as mean 
   s.e . for each construct .
the 
  indicated plasmids expressing various rac1v12 mutants were used to transfect 
  cos1 cells . all constructs except mcfp - rac1(v12 ) were defective in 
  self - association ( figs . 3 and 
  4 ) , whereas only 
  mcfp - rac1(v12)(c189s ) and mcfp - rac1(v12)(6q ) were defective in localizing on 
  the plasma membrane .
24 h after transfection , cells were challenged with 
  yp17/pyope for 30 min , fixed , and then immunostained for extracellular 
  bacteria and internalized bacteria .
uptake efficiency of 50 cfp - positive cells 
  from each of the three coverslips was determined and summarized as mean 
   s.e . for each construct .
control , no plasmid included in 
  transfection . as demonstrated previously , rac1v12 restored significant bacterial uptake 
  in the presence of yope , allowing the effects of mutations to be compared 
  ( fig .
not surprisingly , the rac1v12(c189s ) derivative that 
  lacks gerenylgerenylation , and therefore could not target to the membrane , was 
  unable to bypass yope activity ( fig . 
 
similarly defective was the racv12(6q ) mutant , which also could not 
  restore uptake in the presence of yope 
  ( fig .
8 ; mcfp - rac1v12(6q ) 
  versus mcfp - rac1v12 ; p  0.004 ) .
thus , invasin - mediated 
  uptake required the pbr . to determine if the defect in uptake seen in these 
  mutants was due to an absence of self - association or due to loss of effector 
  interactions , the k186e effector binding mutation 
  ( 34 ) that also lowers rac1 
  self - association ( figs . 1 ( b and 
  g ) and 5(h 
  and i ) )
the effect of this mutation on 
  bacterial uptake was similar to that seen with the 6q mutation , since the two 
  derivatives showed no statistical difference in being able to promote uptake 
  ( fig .
8 ; 
  mcfp - rac1v12(k186e ) versus rac1v12(6q ) ; p = 0.47 ) .
since both the 6q and c189s mutations reduce plasma membrane localization 
  of rac1 , we could not determine if lowered uptake was a result of lowered 
  self - association , effector interactions , or plasma membrane localization 
  ( 14 ) . to address this issue , 
 
uptake was analyzed under conditions in which plasma membrane localization of 
  the mutant rac1 proteins was forced , using the constructs that have a lyn 
  nh2-terminal myristoylation site 
  ( 24 ) .
the addition of the lyn 
  myristoylation site significantly suppressed the defect observed in the c189s 
  mutant ( fig .
8 ; 
  mcfp - racv12(c189s ) versus lyn - mcfp - racv12(c189s ) ; p < 
  0.004 )
. therefore , the loss of uptake that results from lack of prenylation 
  appears to be almost totally due to defective membrane localization , because 
  the lyn construct lacking the prenylation site showed no evidence of 
  self - association ( figs . 
  4e,5d , 
  and 6 ) yet
if the 6q mutation was introduced into the lyn construct 
  ( lyn - rac1v12(6q / c189s ) ) , then uptake was as poor as observed in the construct 
  lacking the lyn site ( fig . 8 ; 
  rac1v12(c189s ) versus lyn - rac1v12(6q / c189s ) ; p < 0.6 )
. 
  the defect in uptake resulting from the 6q mutation was unlikely to be due to 
  poor plasma membrane localization , because the myristoylation site was intact 
  ( fig .
does not appear to be essential for 
  promoting invasin - mediated uptake in this system , and the 6q mutation that 
  replaces the pbr is defective even when plasma membrane localization is 
  forced , the pbr must be required either for recognition by downstream 
  effectors or racgef proteins .
the importance of the latter could not be the 
  cause of the defect in this assay , because rac1v12 derivatives were used , 
  which do not require guanine nucleotide exchange factors for activation .
the 
  importance of pbr recognition by effectors for invasin - dependent uptake is 
  supported by results using the k186e mutant .
therefore , it appears that in 
  this system , the pbr was required for downstream effector recognition .
in this report , we used fret technology to demonstrate that rac1 is able to 
  self - associate in live cells and that the highest levels of this interaction 
  occurred at sites documented to contain the highest concentrations of actin 
  remodeling ( 14 , 
  22 ) , such as regions of cell 
  surface ruffling and nascent phagosome formation during bacterial uptake . 
  these are also the regions of the cell that have the highest levels of 
  activated rac1 ( 14 , 
  22 ) .
this result supports the 
  model that the rac1 pbr is an important determinant of self - association 
  ( 21 ) .
our data indicate that 
  the pbr is not sufficient for this interaction within host cells , however , 
  since insertion of rac1 into the membrane via its caax prenylation 
  motif was required for detectable self - association . using an assay for the 
  ability of rac1 to support uptake of y. pseudotuberculosis
, we 
  demonstrated that rac1 self - association was not required for driving actin 
  polymerization events that lead to internalization of the microorganism .
the 
  pbr , however , was important for efficient uptake in this system under 
  conditions in which uptake did not require self - association of rac1 , 
  indicating that the pbr was probably required for interaction with downstream 
  effectors .
the nature of the assay used to analyze uptake involved using 
  constitutive active derivatives of rac1 and did not rule out the possibility 
  that rac1 self - association may be important to stimulate its activation .
rac1 self - association was originally demonstrated using gel filtration 
  experiments , which showed that the cooh - terminal pbr was required for the 
  formation of high molecular weight species of rac1 that had been purified as a 
  nonprenylated protein ( 21 ) . 
 
our observations are consistent with the importance of this region in 
  promoting self - association within mammalian cells 
  ( fig .
1b ) , but unlike 
  this biochemical assay , we could obtain no evidence for self - association in 
  the absence of membrane localization .
in particular , the orientation of the 
  protein relative to the membrane was extremely important , since forcing 
  membrane localization by an nh2-terminal myristoylation tag was not 
  sufficient to generate a rac1:rac1 fret signal ( figs . 
  4 and 
  5 ) .
it may be that an important 
  nucleation event that initiates self - interaction requires a particular 
  orientation of the protein in the membrane .
alternatively , prenylation could 
  target rac1 into microdomains on the plasma membrane that are required for 
  close association of rac1 monomers .
first , active rac1 is preferentially localized on 
  cholesterol - rich lipid raft domains , which could stimulate self - interaction 
  ( 35 ) .
second , rac1 
  preferentially self - associated at the sites of actin polymerization observed 
  in lamellipodia and nascent phagosomes .
after recruitment to these sites , 
  there could be back - signaling to membrane - localized rac1 that stimulates 
  self - association .
in fact , the presence of a high concentration of active rac1 
  at these sites ( 13 , 
  14 ) could act as a further 
  important stimulus of rac1 self - interaction , perhaps by initiating cycles of 
  signaling from membrane - localized rac1 to downstream effectors and back to 
  membrane - localized rac1 .
the self - association initiated at the host cell membrane is stable in the 
  presence of gentle detergent , but it may have a limited lifetime within a host 
  cell once rac1 is liberated from the membrane .
the fret data obtained in this 
  study could be reproduced using immunoprecipitation analysis , in which 
  epitope - tagged rac1 variants were used to identify self - interaction complexes 
  ( fig .
6 ) . only rac1 derivatives 
  that showed self - association by fret analysis were positive in the 
  immunoprecipitation assay ,
so the fret analysis was a good predictor of 
  whether multimeric rac1 complexes could survive the detergent extraction 
  procedure .
release of rac1 from the membrane by the yersinia yopt 
  protease , which removes the caax prenylation signal , disrupted 
  self - interaction ( fig .
. this 
  could be due to binding of soluble proteins within the cell after release or 
  the introduction of the protein into a microenvironment within the cell that 
  stimulates dissociation .
rhogdi is the most obvious candidate for a protein 
  that could stimulate dissociation of rac1 monomers ; however , the proteolytic 
  product of yopt , nonprenenylated rac1 , is not a good substrate for rhogdi 
  ( 36 ) .
release of rac1 from the 
  membrane by yopt results in pbr - dependent translocation of rac1 into the 
  nucleus , where at least a subpopulation of the protein is active 
  ( 22 ) .
although it is not clear 
  what downstream effectors interact with the protein in this compartment , 
  either the biochemical environment of the nucleus or its interacting partners 
  could interfere with self - association .
the close correlation between results of the fret assay and the 
  co - immunoprecipitation of rac1 derivatives indicated that the fret analysis 
  could be reproduced using traditional methods of detecting interaction .
this 
  connection was not predicted from previously published work using 
  membrane - targeted cfp / yfp derivatives , which indicated that acylated proteins 
  inserted into membranes associate with each other in the absence of any 
  evidence of stable protein interactions 
  ( 24 ) .
( 24 ) 
  showed that the geranylgeranylated myfp / mcfp pair generated a 
  concentration - independent fret signal .
the fret by proximity observed in that 
  work , in the absence of any protein domains that promote interaction , is 
  thought to be due to the formation of microdomains within membranes , resulting 
  in close congregation of proteins 
  ( 24 ) .
although not 
  investigated in that work , it is unlikely that such proximity interactions 
  could have survived detergent extraction and allowed co - immunoprecipitation of 
  protein pairs .
presumably , our fixation procedures prior to fret ( see 
   materials and methods  ) prevented fluorophores that are located 
  on closely oriented but noninteracting proteins from generating a signal , 
  indicating that only strong protein - protein interactions can be detected .
a 
  recent study indicates that fixation can reduce the yield of fret in selected 
  circumstances , particularly when both fluorophores are fused on a single 
  polypeptide .
mathematical modeling indicates that a likely explanation for 
  this reduction is that with some fret pairs , fixation can limit protein 
  flexibility and lock the fluorophores in conformations that interfere with 
  energy transfer ( 38 ) . although 
  such interference appears irrelevant in the case of two physically unlinked 
  proteins that have interacting regions 
  ( 38 ) , it is possible that fret 
  caused by proximally localized but noninteracting proteins requires a level of 
  flexibility of the fluorophore that is blocked by fixation .
it has been suggested that pbr - mediated self - association of rac1 might 
  drive optimal effector signaling , based on the observation that a 
  pbr - defective rac1 derivative was highly defective in activating one of its 
  downstream effectors , the serine / threonine kinase pak1 
  ( 21 ) . arguing against this 
  hypothesis were results from another study showing that 
  nh2-terminal myristoylation could fully restore the ability of the 
  pbr - defective rac1 to activate the kinase activity of pak1 
  ( 13 ) . from our studies , we 
  think it likely that the restoration of pak1 signaling was due to the 
  myristoyl group forcing membrane localization of the rac1 mutant rather than 
  due to self - association .
our results support the idea that the loss of 
  membrane targeting that results from the disruption of the pbr is a more 
  profound consequence than loss of self - association , because membrane targeting 
  of rac1 in the absence of self - association appeared sufficient to promote 
  uptake of y. pseudotuberculosis .
a membrane - targeted myristoylated 
  rac1v12(c189s ) did not self - associate , yet this activated rac1 mutant was able 
  to restore bacterial uptake under conditions in which the endogenous rac1 was 
  inactive , as long as the pbr was intact in such constructs .
in addition to promoting self - association and membrane targeting , the pbr 
  is known to facilitate nuclear localization of rac1 and binding to 
  pip5k as well as other downstream effectors of rac1 
  ( 7 , 
  31 , 
  37 , 
  39 ) .
phosphatidylinositol 
  4,5-bisphosphate , the product of pip5k , stimulates invasin - promoted uptake 
  ( 6 ) .
therefore , the importance 
  of pbr could be explained by its ability to stimulate localized 
  phosphoinositol 4,5-bisphosphate accumulation at the phagocytic cup .
the fact 
  that we could not bypass the requirement for the pbr in bacterial uptake by 
  simply forcing localization of the protein into the membrane 
  ( fig .
8) was very different 
  from the result showing that pak1 signaling was pbr - independent after 
  localization of rac1 in the plasma membrane 
  ( 14 ) .
therefore , stimulation 
  of pak1 is probably not sufficient to bypass the requirement of this sequence 
  determinant for bacterial uptake .
consistent with this hypothesis was the 
  finding that a less drastic change in the pbr that interferes with 
  pip5k binding ( the rac1(k186e ) allele ) , was sufficient to interfere 
  with the ability of a membrane - localized and constitutively active rac1 
  variant to promote uptake .
although the evidence presented here argue that loss of multimerization 
  resulting from alterations in the pbr can not explain the observed defects in 
  invasin - mediated uptake , it should be pointed out that the rac1 variants used 
  to test uptake were activated forms of the protein 
  ( fig .
at least for the 
  integrin receptor , multimerization is important , although this does not mean 
  that downstream signaling molecules require multimerization .
artificially 
  dimerizing invasin greatly reduces the concentration of the protein required 
  to promote uptake of particles 
  ( 19 ) .
the evidence that 
  downstream multimerization my play a role in regulating this event is that 
  many gtpases are known to form oligomers , an event associated with 
  self - stimulatory gap activity 
  ( 21 ) .
although this latter 
  observation argues that multimerization plays a negative role in signaling , 
  self - inhibition of rac1 may provide a mechanism to limit rac1 activation that 
  is important for successful completion of cytoskeletal activities .
such 
  negative feedback may be critical at sites of local rac1 activity , especially 
  when gap and rhogdi activities are expected to be down - regulated . since 
  downstream signaling from rac1 to other gtpases has complex consequences on 
  invasin - mediated uptake ( 23 ) , 
  localized down - modulation of rac1 signaling could play an important role in 
  completing phagocytic events .
future critical tests of the importance of multimerization in either 
  down - modulation of rac1 activity or stimulating downstream signaling will 
  require the development of straightforward systems to analyze mutants at 
  limiting and highly defined protein concentrations .
even so , this work 
  demonstrates that multimerization and membrane localization promoted by the 
  pbr do not encompass the entire spectrum of events that are modulated by this 
  small region of the protein .
 the cooh - terminal polybasic region ( pbr ) of rac1 , a rho family gtpase 
  member , is required for rac1 self - association , membrane localization , nuclear 
  translocation , and interaction with downstream effectors . 
 we previously 
  demonstrated that phosphatidylinositol-4-phosphate 5-kinase , one of the 
  effectors that requires the polybasic region for interaction , is necessary for 
  efficient invasin - promoted uptake of yersinia pseudotuberculosis by 
  nonphagocytic cells . 
 here we further examined the role of this region in 
  invasin - promoted uptake . using fluorescence resonance energy transfer 
  experiments ( fret ) , we determined that engagement of integrin receptors by 
  invasin caused elevated levels of rac1 self - association at the site of 
  bacterial adhesion in a pbr - dependent fashion . 
 self - association could be 
  disrupted using several strategies : translocation of the yersinia 
  yopt prenylcysteine protease into host cells , inactivation of the rac1 
  isoprenylation signal that is required for membrane localization , and 
  elimination of the pbr . 
 disruption in each case impaired invasin - promoted 
  uptake . to determine 
 if there is a role for the pbr in rac1 effector signaling 
  that was independent of its role in membrane localization or multimerization , 
  we examined the effect of the pbr in the context of a rac1 derivative that was 
  targeted to the membrane via an nh2-terminal lipid tail . 
 the 
  membrane - targeted rac1 derivative restored significant invasin - promoted 
  bacterial uptake in a pbr - dependent manner and yet displayed no detectable 
  self - association . 
 this study indicates that , in addition to its role in 
  promoting membrane localization , the pbr exerts a positive effect on 
  rac1-controlled bacterial uptake that is independent of rac1 self - association , 
  most likely due to signaling to downstream effectors . 
MATERIALS AND METHODS
RESULTS
DISCUSSION