mutations were introduced into has by megaprimer mutagenesis ( sarkar   and sommer 1990 ; thomas and roth , 1994 ) . the pcr product was digested with xbai and bamhi and used to replace an xbai to bamhi fragment in an expression vector pcb6-ha - y543 xba that encoded the 133   carboxyl - terminal amino acids of japan ha .
each pcr fragment was sequenced to confirm the identity of the mutation and that second - site mutations had not occurred .
mdck subline d5 cells ( brewer and roth ,   1995 ) were transfected with ha mutants in the expression vector pcb6   ( brewer , 1994 ) and selected for survival in g418 .
the uncloned , drug- resistant cell population was used for experiments to avoid any effects   from clonal variation .
mdck cell monolayers were grown , treated with 10 mm sodium butyrate ,   and then labeled with 1.14 ci / ml transslabel ( icn biomedicals , inc . , irvine , ca ) as described ( lin et al . , 1997 ) .
to measure rates of entry to the   golgi complex , monolayers expressing each mutant ha that had been labeled with radioactive amino acids were chased with prewarmed nonradioactive medium at 37c for either 0 , 10 , 20 , 30 , or 60 min .
the immunoprecipitates were analyzed by page and quantified by scanning with a phosphorimager ( molecular dynamics , inc . ,
the proportion of   each immunoprecipitated protein that had shifted to the slower migrating ,   golgi - modified form was measured as a fraction of the total labeled ha in   the immunoprecipitate . to measure rates of arrival at the cell surface ,
cells were pulse - labeled   as described above and chased at 37c in dme containing 10 g / ml of   trypsin for 0 , 30 , 60 , 120 , or 180 min .
trypsin cleaves ha arriving at the   cell surface into two disulfide - bonded fragments , ha1 and ha2 .
the cells   were shifted to ice and treated with 100g / ml of soybean trypsin inhibitor   ( sti ) for 15 min , and then has were immunoprecipitated , separated by   page , and the amount of ha0 , ha1 , and ha2 was quantified .
for each   sample , the percentage of ha at the cell surface was calculated as ( [ ha1 +   ha2]/ total ha )  100% . for the 4a511 protein , the chase medium did   not contain trypsin and arrival at the cell surface
was measured by biotinylation at 4c as previously described ( brewer and roth , 1991 ) .
these assays were essentially as previously described ( skibbens et al . ,   1989 ; lin et al . , 1997 ) .
for assays of polarized transport , confluent mdck   monolayers expressing each mutant ha were grown for 5 d on filter culture inserts .
cells were treated with dme containing 10 mm sodium butyrate for 15 h to increase ha expression and were pulse labeled as described ( lin et al . , 1997 ) .
for experiments in which cholesterol was   depleted from the cells before the chase , cells were grown for 4 d in dme   containing 10% fetal bovine serum .
16 h before the chase , cells were   treated with dme containing 10% lipoprotein - deficient newborn calf serum and 50 m of compactin ( hua et al . , 1996 ) .
50 m of compactin was   also present in media used for labeling with radioactive amino acids .
after   pulse labeling , has were chased to the cell surface in serum - free dme .  
for each ha , one sample had trypsin present in the apical chase medium ,   one had trypsin in the basolateral chase medium , and one had no trypsin .  
excess sti was always included in the medium on the side of the filter opposite to that containing trypsin . at the end of the chase
, samples were   treated with sti and the has were immunoprecipitated and analyzed as   described for the cell surface assay .
the percentage of ha at the apical   surface was calculated by dividing the fraction of labeled ha that was   cleaved by trypsin at the apical surface by the fraction of labeled ha that   was cleaved at both apical and basolateral surfaces ( the surface population )  100% .
each value was corrected for the fraction of ha1 + ha2   in samples chased in the absence of trypsin , which was usually < 10% . for assays of insolubility in triton x-100
cells were then treated for 15 h with sodium   butyrate , were labeled with 1.14 ci / ml transslabel for 15 min at 37c ,   and then chased in normal medium for 80 min .
cells were extracted on ice   for 30 min in 50 mm tris - hcl , ph 7.5 , containing 150 mm nacl , 2 mm   edta , 2 mm dtt , 1 protein inhibitor set ( boehringer mannheim   biochemicals , inc . , indianapolis , in ) , 100 g / ml sti , and 1% triton   x-100 .
cells were scraped into 1.5-ml microfuge tubes and were centrifuged at 12,000 g for 5 min ( model microfuge r ; beckman coulter , fullerton , ca ) .
pellets   were solubilized with 50 mm tris - hcl , ph 8.8 , containing 5 mm edta   and 1% sds and passed several times through a 27-gauge needle .
the   pellet fraction was diluted with extraction buffer and supernatant fractions with sds so that the final concentration of sds in each was 0.17% .  
mutations were introduced into has by megaprimer mutagenesis ( sarkar   and sommer 1990 ; thomas and roth , 1994 ) . the pcr product was digested with xbai and bamhi and used to replace an xbai to bamhi fragment in an expression vector pcb6-ha - y543 xba that encoded the 133   carboxyl - terminal amino acids of japan ha .
each pcr fragment was sequenced to confirm the identity of the mutation and that second - site mutations had not occurred .
mdck subline d5 cells ( brewer and roth ,   1995 ) were transfected with ha mutants in the expression vector pcb6   ( brewer , 1994 ) and selected for survival in g418 .
the uncloned , drug- resistant cell population was used for experiments to avoid any effects   from clonal variation .
mdck cell monolayers were grown , treated with 10 mm sodium butyrate ,   and then labeled with 1.14 ci / ml transslabel ( icn biomedicals , inc . , irvine , ca ) as described ( lin et al . , 1997 ) .
to measure rates of entry to the   golgi complex , monolayers expressing each mutant ha that had been labeled with radioactive amino acids were chased with prewarmed nonradioactive medium at 37c for either 0 , 10 , 20 , 30 , or 60 min .
the immunoprecipitates were analyzed by page and quantified by scanning with a phosphorimager ( molecular dynamics , inc . ,
the proportion of   each immunoprecipitated protein that had shifted to the slower migrating ,   golgi - modified form was measured as a fraction of the total labeled ha in   the immunoprecipitate . to measure rates of arrival at the cell surface ,
cells were pulse - labeled   as described above and chased at 37c in dme containing 10 g / ml of   trypsin for 0 , 30 , 60 , 120 , or 180 min .
trypsin cleaves ha arriving at the   cell surface into two disulfide - bonded fragments , ha1 and ha2 .
the cells   were shifted to ice and treated with 100g / ml of soybean trypsin inhibitor   ( sti ) for 15 min , and then has were immunoprecipitated , separated by   page , and the amount of ha0 , ha1 , and ha2 was quantified . for each   sample , the percentage of ha at the cell surface was calculated as ( [ ha1 +   ha2]/ total ha )  100% . for the 4a511 protein , the chase medium did   not contain trypsin and arrival at the cell surface
was measured by biotinylation at 4c as previously described ( brewer and roth , 1991 ) .
these assays were essentially as previously described ( skibbens et al . ,   1989 ; lin et al . , 1997 ) . for assays of polarized transport , confluent mdck   monolayers expressing each mutant ha
cells were treated with dme containing 10 mm sodium butyrate for 15 h to increase ha expression and were pulse labeled as described ( lin et al . , 1997 ) .
for experiments in which cholesterol was   depleted from the cells before the chase , cells were grown for 4 d in dme   containing 10% fetal bovine serum .
16 h before the chase , cells were   treated with dme containing 10% lipoprotein - deficient newborn calf serum and 50 m of compactin ( hua et al . , 1996 ) .
50 m of compactin was   also present in media used for labeling with radioactive amino acids .
after   pulse labeling , has were chased to the cell surface in serum - free dme .  
for each ha , one sample had trypsin present in the apical chase medium ,   one had trypsin in the basolateral chase medium , and one had no trypsin .  
excess sti was always included in the medium on the side of the filter opposite to that containing trypsin . at the end of the chase ,
samples were   treated with sti and the has were immunoprecipitated and analyzed as   described for the cell surface assay .
the percentage of ha at the apical   surface was calculated by dividing the fraction of labeled ha that was   cleaved by trypsin at the apical surface by the fraction of labeled ha that   was cleaved at both apical and basolateral surfaces ( the surface population )  100% .
each value was corrected for the fraction of ha1 + ha2   in samples chased in the absence of trypsin , which was usually < 10% . for assays of insolubility in triton x-100
cells were then treated for 15 h with sodium   butyrate , were labeled with 1.14 ci / ml transslabel for 15 min at 37c ,   and then chased in normal medium for 80 min .
cells were extracted on ice   for 30 min in 50 mm tris - hcl , ph 7.5 , containing 150 mm nacl , 2 mm   edta , 2 mm dtt , 1 protein inhibitor set ( boehringer mannheim   biochemicals , inc . , indianapolis , in ) , 100 g / ml sti , and 1% triton   x-100 .
cells were scraped into 1.5-ml microfuge tubes and were centrifuged at 12,000 g for 5 min ( model microfuge r ; beckman coulter , fullerton , ca ) .
pellets   were solubilized with 50 mm tris - hcl , ph 8.8 , containing 5 mm edta   and 1% sds and passed several times through a 27-gauge needle .
the   pellet fraction was diluted with extraction buffer and supernatant fractions with sds so that the final concentration of sds in each was 0.17% .  
to investigate a possible role for the ha tm in apical   transport of ha in mdck cells , a cdna for ha was mutated to produce a series of has in which blocks of four or   five contiguous amino acids were converted to alanines at   different positions throughout the entire tm ( fig .
1 ) . in   addition , since it has been proposed that interactions between lipid head groups might stabilize the association of   lipids in digs ( simons and ikonen , 1997 ) , four amino acids preceding the tm were also changed to alanine .
a   highly polarized mdck clonal cell line , d5 , used previously by us ( thomas and roth , 1994 ; lin et al . , 1997 ) , was   transfected with expression plasmids containing the mutant cdnas and uncloned , polarized mdck cell populations were selected that stably expressed mutant has .   since the ha tm can influence the folding and trimerization of the protein ( lazarovits et al . , 1990 ) , and trimerization
is required for efficient export of ha from the er   ( copeland et al . , 1986
; gething et al . , 1986 ) , the rate at   which each protein reached the golgi complex or cell surface was measured by pulse - chase experiments ( table i ) .   only one of the mutant proteins , ha 2a514 , ( fig .
1 ) , was   found to be severely defective in transport to the golgi   complex .
the other proteins were exported from the er   to the golgi complex at rates that ranged from 50 to 100%   of the rate of the wild - type ha .   
properly folded ha is cleaved by extracellular trypsin at   a single site , producing mature , biologically active ha   composed of disulfide - linked ha1 and ha2 subunits   ( wiley and skehel , 1987 ) .
when pulse - labeled mutant   has were chased in medium containing trypsin , only ha   4a511 was degraded by the trypsin , indicating that it was   not properly folded .
the other mutant proteins were   cleaved into ha1 and ha2 and reached the cell surface as   fast or faster than wild - type ha , and to a comparable extent , with the exception of ha 2a514 which was retained   in the er .
mdck cells expressing each of the ha mutants or the   wild - type ha were grown as confluent monolayers on filter culture inserts , and the ability of the cells to sort ha   into the apical pathway was determined by pulse - chase   protocols ( matlin and simons , 1984 ; brewer and roth ,   1991 ) .
an image of a typical experiment is shown in fig . 2 a. we estimate from labeling experiments such as this that   expression of the various mutant has differed by no more   than fourfold and in each case was relatively low , at least   50-fold less than in mdck cells infected with influenza virus ( data not shown ) .
no correlation between sorting of   has and expression level in these cell lines was observed .   the results of many experiments measuring delivery of   has to the apical and basolateral surfaces of mdck   monolayers grown on filter inserts are presented in table   ii . changing the last five amino acids of the ha tm sequence to alanines ( 5a531 )
changing sequences of   four or five amino acids to alanines on the other side of the   transmembrane domain , either before ( 4a507 ) or immediately after the point where the chain is predicted to enter   the outer bilayer ( 2a511 , 4a511 ) caused the protein to be   delivered equally to the apical and basolateral surface .  
however , mutation of residues predicted to lie near the   base of the outer leaflet of the bilayer had a much more   dramatic effect .
this is seen most clearly   with the 2a520 protein , which was sorted more efficiently   to the basolateral surface than the wild - type ha was   sorted to the apical .
thus , there is a critically important region near the center of the ha tm that is necessary for   the protein to enter into apical transport pathways ( table   ii ) and mutation of this region did not affect the folding of   the external domain of ha ( table i ) .
mutation of residues   immediately beyond position 520 , which would be predicted to lie within the inner leaflet of the bilayer , also prevented apical sorting but were less severe .    to investigate the individual contributions for apical  
sorting of residues g520 and s521 of the a / japan/305 ha ,   each was mutated in separate cdnas .
mutation of   s521 to alanine produced as severe a defect as deleting   both g520 and s521 , and caused the s521a protein to be   delivered predominately to the basolateral surface .
scheiffele and colleagues ( 1997 ) have shown that the   ability of the ha to associate stably with digs in fibroblasts is sensitive to changes in the sequences of the portion of the tm that span the outer leaflet of the bilayer .  
the sequences that would span the inner leaflet were   found to be relatively unimportant for this property .
we   confirmed these observations in mdck cells expressing   the ha tm mutants ( figure 2 b and table ii ) .
consistent   with the hypothesis that association with detergent - insoluble membrane domains is required for proper sorting of   ha into an apical transport pathway , we did not observe   proper sorting of any mutant protein that was soluble in   triton x-100 .
however , mutants 5a528 , a516s , and   g520s were as insoluble in triton x-100 as was wild - type   ha , but were not sorted properly to the apical surface .  
thus , association of ha with digs appears to be necessary , but not sufficient , for apical sorting .
the observation that the 2a520 mutant was transported   predominately to the basolateral surface of mdck cells   could be explained either by exclusion of most of the mutant protein from apical transport pathways , forcing it into   the basolateral pathway , or by creation of a positive basolateral signal that allowed active basolateral sorting .
there   has been no previous evidence for either a cryptic basolateral sorting signal in ha or for the existence of basolateral   sorting signals in tm domains
. however , there is evidence   that efficient basolateral sorting of the na / k atpase   requires an intact apical pathway containing digs , which   presumably excludes the na / k atpase and forces it   into the basolateral pathway ( mays et al . , 1995 ) .
if basolateral sorting of the 2a520 mutant occurred by a similar   mechanism , then treatments that inhibited the formation   of digs might allow ha 2a520 access into apically directed vesicles , and the protein should be exported randomly .
digs require cholesterol to form ( schroeder et al . ,   1994 ; scheiffele et al . , 1997 ) .
thus , mdck cells expressing   either the ha 2a520 mutant , wild - type ha , or ha+8   d549h , which contains a dominant basolateral signal ( lin   et al . ,
1997 ) , were grown as monolayers and depleted of   cholesterol . under the conditions used ,
the ability of ha   to partition into membranes insoluble in triton x-100 was   decreased by 50% .
the delivery of each of these has to   the plasma membranes of cells depleted in cholesterol was   measured ( table iii ) .
depletion of cholesterol had no effect on the apical transport of wild - type ha , indicating   that the apical pathway functioned under these conditions .   however , twice as much ha 2a520 reached the apical   surface in cholesterol - depleted cells as did when cholesterol levels were normal , resulting in randomized transport of ha 2a520 .
this suggests that the presence of   digs in cells having normal levels of cholesterol excluded   this mutant from apical pathways .
cholesterol depletion   had no effect on the basolateral pathway , as shown by continued basolateral transport of ha+8 d549h .
the effects of mutations in the transmembrane domain of   the a / japan ha are summarized in fig .
3 . residues i514   and y515 were critical for proper folding of this ha .
although there was some reduction in transport rates observed for mutants with changes in other positions , the   folding and trimerization of ha was generally tolerant of   mutation of other tm residues to alanine .
mutations in   the region of ha predicted to be just outside and within   the outer leaflet of the bilayer caused a loss of apical sorting and loss of insolubility in triton x-100 .
the effects of the   mutations increased as the region mutated approached   two tm residues at positions 520 and 521 that are highly   conserved among influenza virus ha types ( fig .
4 ) , and   decreased as the region mutated approached the portion   of the tm that would reside in the inner leaflet of the bilayer .
the pattern observed is consistent with ha having   an important recognition feature within the membrane   that contains s521 as the most important element .   
the observations that certain ha mutants not only lost   the ability to be sorted , but were actually sorted to the basolateral surface as efficiently as the wild - type ha was   sorted apically , could have two independent causes .
either   ha contains weak basolateral sorting information that is   normally recessive to apical sorting information in the tm ,   which the mutant lacks , or ha must be able to enter digs   to gain access to the apical surface , and the mutant can not   enter digs .
our data support the latter interpretation .   decreasing cholesterol content in cells to the extent that   twofold less
ha partitioned into digs had no effect on   sorting of wild - type ha , or a mutant of ha with a strong   basolateral sorting signal . however , transport of the   2a520 mutant became random under the same conditions .  
these results suggest that the 2a520 mutant had no basolateral sorting information , but in the presence of digs   was excluded from the apical pathway . under the conditions of our experiments , lowering cholesterol levels such that half as much
wild - type ha was recovered in a cell fraction insoluble in triton x-100 did not   decrease sorting of ha to the apical surface . recently ,  
keller and simons ( 1998 ) reported that depleting cholesterol levels by 6070% caused missorting of ha to the basolateral surface of mdck cells infected with influenza virus .
however , in our cell lines , ha expression is at least   50-fold lower than in cells infected with influenza virus   and it is possible that sufficient apical sorting capacity remained intact under our experimental conditions to accommodate the available ha cargo . nonetheless , the fact   that only half as much wild - type ha was found in the detergent - insoluble fraction but apical sorting was unchanged means that the sorting event is not identical to the   partitioning event , as measured by this assay . in cholesterol - depleted cells ,
either the apical sorting machinery remained with the residual digs and was sufficient to sort   has , or the machinery was able to interact with the apical   signal in the tm of has outside of digs .
the later possibility is supported by the observation that mutant a516s   was as insoluble in triton x-100 as was wild - type ha , but   was not sorted apically .
thus , entry into detergent insoluble membranes is not sufficient for apical sorting of ha .
a simple explanation for our observations is that apical   sorting machinery is normally sequestered in digs .
we   propose that sequences including s521 form the binding   site for an integral membrane protein associated with   digs .
the behavior of the ha tm mutants is   consistent with a multistep process for sorting proteins   into the apical pathway . in the first step ,
only proteins capable of entering membrane domains enriched in glycosphingolipids are able to come into contact with apical   sorting machinery associated with those domains .
however , only those proteins having the ability to bind tightly   to elements of the sorting apparatus will be efficiently concentrated within the digs and be effectively sorted to the   apical surface . the degree to which transmembrane proteins lacking sorting signals would reach the apical surface   would depend upon their partitioning coefficient with   digs , and upon the availability of empty space within the   nascent apical transport vesicle . the latter would depend   upon the concentration of cargo with apical sorting signals .  
all   evidence suggests that the basolateral sorting machinery   resides outside of digs . the probability that a protein   would be transported basolaterally would depend upon   the relative affinity of that protein for the basolateral sorting machinery , its partitioning coefficient with digs , and   available space within the basolateral vesicles .
our observations with the ha mutant 2a520 indicate that , under   certain conditions , the inability to partition into digs may   be sufficient to force a protein into the basolateral pathway .
perhaps the converse is true , that efficient partitioning into digs might keep proteins from entering the basolateral pathway by default .
it is possible that proteins with   glycosphingolipid membrane anchors are sorted apically   in such a manner , without interacting with the machinery   that sorts apical transmembrane proteins .
amino acids of the a / japan   h2 ha tm are given in single letter code beginning with the last   four residues of the external domain and ending with the residue   predicted to be the last of the tm .  , no change from the wild - type sequence ;  , deletion of the residue at that position .
effect of mutations in the ha tm on kinetics of   transport through the secretory pathway   results are shown from a representative pulse - chase experiment on mdck cells permanently expressing various ha mutants .
arrival at the golgi was monitored by the   appearance of terminally glycosylated ha , which migrates more slowly during page .  
arrival at the cell surface was measured as the percentage of ha that was converted   into its ha1 and ha2 subunits by trypsin added to the chase medium .
mutations in the ha tm affect transport to the apical   cell surface and solubility in triton x-100 . ( a ) mdck monolayers expressing the ha types shown were grown on filter culture   inserts for 5 d and then subjected to a pulse - chase protocol in   which trypsin was present during the chase in either the apical   ( ap ) or basolateral ( bl ) compartment .
after the chase , has   were immunoprecipitated , separated by page , and then analyzed by a phosphorimager . a representative image comparing   ha and two mutants is shown .
( b ) mdck cells expressing the   mutants shown were pulse - labeled and chased for 40 min , then   the cells were lysed in 1% triton x-100 on ice .
the cell lysate was   centrifuged in a microfuge and separated into supernatant ( s )   and pellet ( p ) fractions .
ha was immunoprecipitated from each   fraction and analyzed as in a. effect of mutations in the ha transmembrane   domain on the delivery of the protein to the apical surface of   mdck cells   results are shown from pulse - chase experiments on mdck cells permanently expressing various ha mutants .
arrival at the apical cell surface was measured by adding trypsin to the chase medium on either the apical or basolateral side of the cell to   convert ha into its ha1 and ha2 subunits .
the percentage of ha arriving at the apical surface was calculated by dividing fraction of ha cleaved to ha1 and ha2 when   trypsin was present at the apical side of the monolayer by the fraction cleaved by   trypsin present on both sides of the monolayer .
values are averages from three or   more independent experiments and are presented with standard deviations .
the fraction of each mutant that was insoluble when cells were lysed in triton x-100 was determined in parallel experiments . for three or more experiments
depletion of cellular cholesterol allows expression   of ha 2a520 to become more apical but has no effect on the   distribution of wild - type ha or ha+8 d549h , a   basolaterally expressed ha mutant *   cholesterol was depleted from mdck cell monolayers expressing each of the ha   proteins by treatment overnight in cholesterol - free medium containing 50 m of compactin according to published methods ( hua et al . , 1996 ) .
distribution of has at the   cell surface was measured as described for table ii .
the   thickness of the bar beneath the ha tm sequence is proportional   to the severity of the inhibition of apical transport .
* , positions of   the sequence most conserved among influenza a and b has .
transmembrane sequences from 36 ha proteins selected from all 14 influenza a subtypes and including three type b has were compared .   for each position in the sequence of the tm ,
a conservation   value was calculated by dividing the percentage of sequences having the most common amino acid by the number of different   amino acids found at that position .
for each position in the tm   sequence , the conservation value and the most common amino   acid are displayed .
amino   acids that would reside in the outer leaflet of the membrane bilayer are more conserved than those predicted to reside within   the inner leaflet .
 the composition of the plasma membrane   domains of epithelial cells is maintained by biosynthetic   pathways that can sort both proteins and lipids into   transport vesicles destined for either the apical or basolateral surface . in mdck cells , 
 the influenza virus hemagglutinin is sorted in the trans - golgi network into   detergent - insoluble , glycosphingolipid - enriched membrane domains that are proposed to be necessary for   sorting hemagglutinin to the apical cell surface . 
 site- directed mutagenesis of the hemagglutinin transmembrane domain was used to test this proposal . 
 the region   of the transmembrane domain required for apical   transport included the residues most conserved among   hemagglutinin subtypes . 
 several mutants were found to   enter detergent - insoluble membranes but were not   properly sorted . 
 replacement of transmembrane residues 520 and 521 with alanines converted the 2a520   mutant hemagglutinin into a basolateral protein . depleting cell cholesterol reduced the ability of wild - type   hemagglutinin to partition into detergent - insoluble   membranes but had no effect on apical or basolateral   sorting . 
 in contrast , cholesterol depletion allowed random transport of the 2a520 mutant . 
 the mutant appeared to lack sorting information but was prevented   from reaching the apical surface when detergent - insoluble membranes were present . 
 apical sorting of hemagglutinin may require binding of either protein or   lipids at the middle of the transmembrane domain and   this normally occurs in detergent - insoluble membrane   domains . 
 entry into these domains appears necessary ,   but not sufficient , for apical sorting . 
Materials and Methods
Cell Lines Expressing Mutant HAs
Folding Assay for HA TM Mutants
Assays for Polarized Transport and Insolubility in  Triton X-100
Results
Discussion
Figures and Tables