parkinson 's disease ( pd ) is a fatal and incurable neurodegenerative disorder characterized by resting tremors , postural instability , and bradykinesia .
the death of midbrain substantia nigra neurons characterizes the pathology of both familial and sporadic forms of pd .
two types of molecules accumulate within these dying cells : misfolded and aggregated -synuclein in cytoplasmic inclusions called lewy bodies   and oxidative free radicals [ 1 , 3 ] .
therefore , a major hypothesis is that -synuclein misfolding and aggregation is linked to neuronal toxicity in pd [ 46 ] .
while the molecular basis of sporadic pd is still unclear , mutations in six genes cause familial pd , including three point mutations ( a53 t , a30p , and e46k ) within the -synuclein gene [ 810 ] .  
in addition to self - aggregation [ 1113 ] , several other -synuclein properties that may be relevant to pd include maintenance of neurotransmitter vesicular pools [ 14 , 15 ] , synaptic plasticity [ 16 , 17 ] , phospholipid binding and lipid metabolism [ 1821 ] , microtubule binding , chaperone - like ability [ 2325 ] , and er - golgi trafficking [ 26 , 27 ] .
while the a30p and a53 t familial mutants are widely studied in how they alter the above properties to cause cytotoxicity and contribute to pd pathogenesis , less is known about the third and most recently discovered familial mutant , e46k .  
thus far , e46k is known to aggregate into fibrils more rapidly than wild - type -synuclein in vitro [ 29 , 30 ] and in cell culture . additionally , recent biochemical studies suggest that e46k enhances contact between the amino and carboxyl domains although whether this interaction increases the likelihood of aggregation is still being sorted .
in contrast to the a30p and a53 t familial mutants , e46k exhibits a reduced accumulation of early oligomers [ 13 , 33 , 34 ] . like wild - type -synuclein , e46k can form ion channel - like pores in lipid bilayers   although recent work suggests that these channels are less conductive than those formed by wild - type -synuclein . on the other hand ,
e46k binds phospholipids more readily than wild - type -synuclein [ 29 , 36 , 37 ] and is more potent in inducing toxicity in neuroblastoma cells that are cotreated with cytokines . in living cells
, however , little is still known of e46k 's ability to bind either the plasma membrane or internal membrane systems . nor is it clear how e46k aggregation and/or membrane association might cause cellular toxicity . budding and fission yeast models have advanced our molecular understanding of several neurodegenerative diseases , including pd [ 21 , 3942 ] .
yeasts revealed that -synuclein binds plasma membranes extensively in addition to its propensity to aggregate , but the relative importance of aggregation and membrane binding to toxicity is still unresolved [ 21 , 4346 ] .
some models demonstrate -synuclein expression and dose - dependent aggregation itself causes toxicity [ 21 , 44 ] , while others report that -synuclein - dependent toxicity , especially when moderately expressed , requires additional factors , such as proteasomal inhibition and oxidative stress [ 43 , 45 ] , or tau coexpression . in 2003 , willingham et al .
similarly , disruption of the secretory pathway   and er - golgi trafficking [ 26 , 27 ] were first found in yeast to increase -synuclein toxicity .   here
, we exploited the utility of both budding yeast and fission yeasts to test the hypothesis that the e46k mutant aggregates and binds membranes more strongly than wt -synuclein in living organisms and that it accumulates to induce cellular toxicity .
indeed , we found that e46k binds the plasma membrane in budding yeast and associates with endomembrane systems and form intracellular aggregations in fission yeast .
while e46k is not toxic to budding yeast , it is toxic to fission yeast especially when associated with internal membrane systems .
as described in our past studies [ 45 , 46 ] , we tagged -synuclein with gfp at its c - terminus and induced its expression with galactose in budding yeast and by removal of thiamine ( the repressor ) in fission yeast .
our first goal was to assess whether e46k was toxic to budding and fission yeasts , and we report that e46k is toxic to yeasts , albeit in a species- and strain - specific manner .
first , for budding yeast , we examined two haploid strains of opposite mating types ( by4741 mat a and by4742 mat  ) and a diploid strain ( by4743 mat a/ ) .
e46k was not toxic to any strain , as assessed by two assays : serial dilution spotting on solid growth plates ( figure 1(a ) ; by4742 not shown ) and growth curves from liquid culture ( data not shown ) .
by4741 cells that expressed e46k grew equally well as cells that did not express -synuclein ( vector alone and gfp ) and those that expressed wt -synuclein or two other familial mutants ( a30p and a53 t ; figure 1(a ) , top ) .
therefore , as we had previously reported for wt , a30p , and a53 t , e46k -synuclein did not adversely affect budding yeast growth .
additionally , neither the mating type ( data not shown ) nor ploidy doubling ( figure 1(a ) , bottom ) altered the lack of e46k -synuclein - dependent toxicity in budding yeast .
next , we examined how -synuclein affected the growth of two haploid fission yeast strains of opposite mating types : h ( tcp1 ) and h+ ( sp3 ) .
in contrast to our budding yeast findings , we found that several -synuclein variants were toxic to fission yeast ( figure 1(b ) ) .
firstly , when compared to vector alone and gfp expressing cells , wt , e46k and a53 t -synuclein expressing cells were each toxic in both strains , but not a30p ( figure 1(b ) ) . in tcp1 , e46k and
wt were toxic to similar extents but less than a53 t ( figure 1(b ) , top ) .
in sp3 , e46k was more toxic than wt yet still less than a53 t ( figure 1(b ) , bottom ) . to gain insight into e46k -synuclein 's differential toxicity in yeasts
e46k -synuclein consistently localized to the plasma membrane of by4741 yeast during 48 hours of expression , similar to wt and a53 t -synuclein localization patterns ( figure 2(a ) ) that we previously had reported for by4741 .
quantification of localization in fluorescing cells indicates that more than 90% expressed e46k -synuclein at the plasma membrane ( figure 2(a ) ) . unlike a53 t
, however , e46k -synuclein was rarely seen aggregated in the cytoplasm ( figure 2(a ) ) . nor did we observe any e46k localization to internal endomembrane systems .
in contrast , a30p -synuclein was mostly cytoplasmically diffuse , similar to gfp alone ( figure 2(a ) ) .
e46k plasma membrane association and the patterns of localization of the other -synuclein variants were maintained in by4742 ( figure 2(b ) ) and by4743 ( figure 2(c ) ) strains .
we next wondered if e46k bound the plasma membrane more extensively than wt -synuclein but found no difference in the extent of plasma membrane association between them over the first 24 hours of expression : both appeared at the plasma membrane as early as 6 hours to similar extents ( figure 2(d ) ) .  
thus , the lack of e46k -synuclein toxicity in budding yeasts correlated with plasma membrane association . in stark contrast to budding yeast , e46k -synuclein was prominently absent at the plasma membrane when examined in the two haploid fission yeast strains ( figure 3 ) . in tcp1 ,
e46k aggregated in the cytoplasm , again strikingly similar to wt and a53 t -synuclein ( figure 3(a ) ) that we previously had reported in tcp1 .
-synuclein and gfp alone were cytoplasmically diffuse ( figure 3(a ) ) , also as previously reported .
most cells had 13 aggregates , but some had up as many as 510 per cell ( figure 3(c ) ) .
these aggregates were outside the nucleus , as determined by dapi staining ( figure 3(d ) , top ) .
notably , dapi staining typically altered the pattern of -synuclein localization , reducing the size and location of -synuclein puncta , which suggests that some of the larger accumulations were either a collection of dispersible smaller aggregates or compact membranous vesicles ( figure 3(d ) , top ) .
initially , up to 24 hours of expression , both e46k and wt -synuclein distributed to the membrane linings of many intracellular compartments , in addition to aggregating in the cytoplasm ( figure 3(b ) ) . over the next 24 hours ,
76% wt -synuclein expressing cells contained aggregates similar to tcp1 , while e46k -synuclein was even more prominently localized to diverse intracellular locations ( 98% cells ) , with no cells containing just aggregates ( figure 3(b ) ) .
over this same course of time , a30p -synuclein remained cytoplasmically diffuse while , a53 t was mostly aggregated by 48 hours ( 84% ) , with endomembrane localization less extensive than e46k ( 16% ; figure 3(b ) ) .
this e46k intracellular localization pattern excluded the nucleus but not the nuclear envelope ( figure 3(d ) , lower ) , and prominently lit up perinuclear and vacuolar / prevacuolar compartments ( figure 3(b ) ) , and other components of the endomembrane system that need further identification . often , vesicular accumulations were observed within cells : mostly with e46k , less with wt , and rarely with a53 t ( figure 3(e ) ) .
thus , the added toxicity of e46k -synuclein in sp3 fission yeast appears correlated best with its localization to diverse endomembrane organelles .  
we previously reported that wt and a53 t -synuclein aggregate in a time- and concentration - dependent manner in fission yeast , providing live cell support for the nucleation polymerization hypothesis . whether e46k does the same has not been reported .
indeed , we found that e46k -synuclein localization in tcp1 was time - dependent ( figure 4(a ) ) . over a time course of 48 hours , e46k was initially diffuse in the cytoplasm for the first 12 hours , but thereafter aggregated into one or more punctate aggregates within individual cells ( figure 4(a ) ) . by 18 hours , less than 5% cells had cytoplasmically diffuse e46k while , wt -synuclein was cytoplasmically diffuse in 10% cells even at 30 hours ( figure 4(a ) ) .  
importantly , the e46k pattern of aggregation closely resembled a53 t -synuclein aggregation ( figure 4(a ) ) .
remarkably , at no point over the time course was e46k -synuclein ( nor wt or a53 t ) localized at the plasma membrane ( figure 3(a ) ) .
a30p -synuclein remained cytoplasmically diffuse and never aggregated over this same time course ( data not shown ) .
similarly , e46k -synuclein also aggregated in tcp1 in a concentration dependent manner , which was demonstrated by growing e46k cells in the presence of decreasing amounts of the repressor , thiamine ( 10 , 1.0 , 0.1 , and 0  m ; figure 4(b ) , left ) . at the two highest thiamine concentrations
, e46k -synuclein was diffusely localized in the cytoplasm ( figure 4(b ) , left ) . however , at the lower thiamine concentrations as more protein is expressed , e46k started aggregating in most cells , similar to wt and a53 t -synuclein ( figure 4(b ) , left ) .
the low level of -synuclein observed by gfp fluorescence even at the highest thiamine concentration was confirmed by western blotting ( figure 4(c ) , right ) .
thus , e46k -synuclein aggregated in a time- and concentration - dependent manner , supporting the nucleation polymerization model .
alternatively , the data can also be explained by concentration - dependent membrane localization as well , if the e46k puncta are , in fact , compact membrane structures , rather than aggregates .
we wondered if the strain - specific enhancement of e46k -synuclein toxicity in fission yeast that was lacking in budding yeast was partly due to its selective accumulation in fission yeast . in budding yeast , e46k and
wt -synuclein were indeed either similar ( by4741 ) or even lower ( by4743 ) at 24 and 48 hours of expression , indicating a lack of accumulation ( figure 5(a ) ) . in fission yeast , however
, we did observe a slight increase in e46k -synuclein expression relative to wt in both tcp1 ( 48  hrs ) and sp3 strains ( both 24 and 48  hrs ; figure 5(b ) ) .
given that serine phosphorylation is linked to -synuclein related pd pathology [ 4850 ] , we next asked if e46k toxicity is linked to phosphorylation .
firstly , we found that both e46k and wt -synuclein were phosphorylated at serine-129 in both budding and fission yeasts and to the same extent ( figure 5(c ) ) .
importantly , the enhanced e46k toxicity in sp3 was not correlated with altered level of -synuclein phosphorylation ( figure 5(c ) ) .  
finally , we asked if the selective e46k -synuclein toxicity in fission yeast was linked to a reduction in organismal growth and survival .
as shown in the schematic ( figure 6(a ) ) , 1000 yeast cells each expressing wt or e46k -synuclein or containing parent vector alone for either 8 ( early log phase of growth ) or 18 hours ( late log phase ) were plated onto repressing or inducing media .
these survival plates were then first grown for two days , and the induced plates were grown even further for three more days .  
firstly , in the tcp1 strain both e46k and wt -synuclein expressing cells were significantly reduced in colony size compared to parent vector cells when they continued to express -synuclein ( induction = figure 6(b ) ) , but not when they stopped expressing -synuclein ( repression  figure 6(b ) ) .
this was true irrespective of whether -synuclein expressing cells were plated from either early or late log phase of growth .
three days later , both wt and e46k -synuclein cells did gain the original colony sizes initially seen with parent vector cells ( figure 6(b ) ) .
we did not observe a reliable difference in colony number between cells that contained parent vector and cells expressing wt or e46k -synuclein ( data not shown ) . in the sp3 strain , where e46k
was shown to be more toxic than wt -synuclein , e46k -synuclein expressing cells formed even smaller colonies than wt -synuclein expressing cells when -synuclein was still being expressed ( induction  figure 6(c ) ) . unlike in tcp1
, these e46k colonies did not reach the colony size of parent vector cells even after three days of addition growth even though wt colonies did ( figure 6(c ) ) . once again
, we did not observe a reliable difference in colony number between cells that contained parent vector and expressing wt or e46k -synuclein ( data not shown ) . in by4741
budding yeast , similar survival assays did not demonstrate any difference in colony size or number between parent vector , wt , and e46k expressing cells under induced conditions at either two or five days of growth ( figure 6(d ) and data not shown ) .
the initial difference in colony size of all yeast cells between repressed ( glucose ) and induced ( galactose ) plates was simply due to the slight general lag in yeast growth in galactose compared to glucose .
thus , the wt and e46k -synuclein toxicity in tcp1 and the additional e46k selective toxicity in sp3 both correlated well with reduced yeast growth but not colony number .
e46k is the most recently discovered of the three -synuclein point mutants that cause familial pd . since its discovery in 2004 , only a handful of mostly in vitro studies
we demonstrate significant organismal evidence that may help explain its toxic potential in pd , and we discuss three notable findings below .
( 1 ) e46k extensively associates with membrane systems in both yeasts but does so in distinct ways : binding plasma membrane in budding yeast and endomembranes in fission yeast .
( 2 ) e46k aggregates in fission yeast , but not in budding yeast , in a time- and concentration - dependent manner . ( 3 ) e46k is toxic to fission yeast in a strain - specific manner that correlates with reduced cellular growth , survival , and modest accumulation . in support of our hypothesis ,
live cell gfp microscopy illustrates that e46k associates with the plasma membrane of budding yeast .
thus , this study is the first to demonstrate e46k association with diverse membrane compartments in living cells , supporting its well - documented affinity for membrane phospholipids in vitro .
since e46k binds liposomes more readily than wt , a30p , or a53 t , and solution nmr work also suggests that e46k adopts a structure that favors lipid binding more rapidly than these other -synuclein variants [ 29 , 37 ] , these studies provide a molecular explanation for why e46k ( more than wt or a53 t ) strongly associates with endomembrane systems in yeasts .
e46k is clearly different from the a30p mutant , which does not associate with membranes within either yeast this study ; [ 45 , 46 ] nor bind lipids in vitro [ 14 , 5153 ] . since e46k enhances n- to c - terminal contacts within -synuclein domains   and is within the n - terminal ktkegv amino acid repeats essential for membrane association [ 10 , 30 , 54 ] , our data strengthens the notion that the e46k impact on the n to c - terminal contact likely facilitates a helical conformation , the structural form required for -synuclein to interact with lipids [ 18 , 55 ] . 

in addition to cytoplasmic localization in neuronal tissue , -synuclein also associates with the secretory pathway [ 56 , 57 ] , localizes to the plasma membrane [ 16 , 5862 ] , and outside cells [ 6165 ] .
thus , e46k 's association ( and to a lesser degree , wt and a53 t ) with internal membrane systems in fission yeast cells is noteworthy .
thus , e46k -synuclein 's prominent presence in perinuclear compartments suggests that it may associate with er and disrupt er to golgi transport to generate toxicity , just as wt and a53 t -synuclein have been reported to do in budding yeast cells [ 26 , 27 ] in budding yeast , overexpression of the secretory pathway specific g protein rab1 also rescues yeasts from -synuclein - dependent [ 26 , 27 ] . while we do not know why e46k ( and to a lesser extent , wt and a53 t ) binds such distinct membrane compartments between the two yeasts , differences in -synuclein concentration in the two yeasts are unlikely to be a major reason , since e46k never associates significantly with the fission yeast plasma membrane even when expressed at low concentrations .
since the two yeast species are separated by almost 400 million years of evolution , inevitable differences in plasma membrane and endomembrane composition are a more likely explanation .
we speculate that differences in the plasma and endomembrane composition exist between budding yeast and fission yeast and that e46k -synuclein might have increased affinity for plasma membrane phospholipids in budding yeast and endomembranes in fission yeast . budding and fission yeasts do have modest differences : in phospholipid composition , in fission yeast being an inositol auxotroph , and fission yeast 's lipid composition varies with temperature and growth .
specific differences between the yeasts may also exist in the composition of membrane proteins with which -synuclein variants interact .
e46k aggregates more rapidly than wt -synuclein in fission yeast , again supporting our hypothesis .
that this , e46k aggregation more closely matches a53 t -synuclein is supported by in vitro evidence that e46k and a53 t aggregate at similar rates [ 29 , 30 ] in cell culture , e46k readily forms intracellular aggregates .
our work supports the notion that e46k enhances -synuclein aggregation due to its location within the n - terminal ktkegv repeats , which influence aggregation rates . synthetic e(xx)k mutations at these points decreased fibrillization lag time in vitro .
rospigliosi et al . also demonstrated that the e46k mutant resulted in n to nac region contact , which , compounded by an overall decrease in negative charge , is hypothesized to increase aggregation rates , providing an explanation for e46k 's impact on aggregation .
the absence of e46k aggregation in budding yeast may be due to two factors : the moderate expression of -synuclein in our lab and the membrane composition differences between the two yeasts . in budding and fission yeasts ,
other labs that express -synuclein at moderate levels also report plasma membrane association [ 42 , 44 ] .
e46k 's enhanced toxicity in fission yeast supports our hypothesis that it is more toxic than wt -synuclein even though neither of them is toxic in budding yeast .
the lack of -synuclein toxicity in budding yeast strain by4741 could be due to dosage - dependent and strain - specific toxicity , as -synuclein is toxic in the w303 strain with two copies expressed but not with one copy .
our fission yeast data indicates that endomembrane association is a key contributor to toxicity although we do not exclude toxicity contributions from interactions with the plasma membrane or cytoplasmic aggregation .
one of the most important questions in pd is the identification of the toxic -synuclein species , and protofibrils possess several features in vitro that make them excellent candidates for being this toxic species , including the ability to permeabilize synthetic vesicles .
both a30p and a53 t form more protofibrils than wt -synuclein in vitro [ 4 , 11 , 73 ] , but little still is known whether e46k does the same , in vitro or in organisms .
we speculate that the differential toxicity ( even within fission yeast strains ) may be due to how much e46k -synuclein protofibrils form within each strain and that endomembrane association may be a significant contributor .
the degree and range of e46k association with endomembrane systems may determine how much it contributes to cytotoxicity .
even though e46k localizes more to internal organelles in tcp1 than wt -synuclein does , their level of toxicity is similar .
we speculate that the added toxicity in sp3 may partly be due to more e46k localization to vacuoles / prevacuoles .
thus , disruption of vesicle integrity may mediate toxicity as much as added association with the er . strain differences in lipid composition may also underlie why sp3 differs from tcp1 in the extent of -synuclein association with intracellular organelles .
since most tcp1 cells express e46k in aggregates , the few cells that do exhibit endomembrane localization likely produce too little toxicity to be detected by the growth and survival assays .
lastly , the difference in toxicity could be due to the h ( tcp1 ) and h+ ( sp3 ) mating - type differences , including altered interactions of -synuclein with secretion of specific mating factors .  
our data confirms an initial report in budding yeast   and extends it to fission yeast that yeasts possess protein kinases that can phosphorylate human -synuclein .
we are also the first to report that wt and e46k -synuclein are phosphorylated in a fission yeast model ( for fuller description , see ) .
although the extent of serine-129 phosphorylation does not correlate with enhanced e46k -synuclein toxicity in fission yeast , it provided us the opportunity to investigate the contribution of phosphorylation to -synuclein aggregation and membrane association in both models that we report elsewhere .
future studies in fission yeast will determine whether -synuclein will disrupt vesicular pool distribution , accumulates in vesicles , and colocalizes with secretory pathway proteins , as it does in budding yeast [ 21 , 27 , 76 ] .
it is possible that such -synuclein endomembrane association also impairs -synuclein degradation and toxicity via lysosmal pathways , as has been observed in budding yeast ( [ 47 , 77 ] , perez et al .
both yeast models recapitulate and extend our in vitro and cell culture knowledge of e46k -synuclein familial mutant properties in two organismal model systems .
together , they illustrate that e46k can both aggregate within cells and associate with its diverse membrane systems .
importantly , the organismal context regulates both these features by varying the extent of aggregation and the extent of association with plasma membrane and endomembrane systems .
our data indicates that -synuclein binding to endomembranes may be key to generating toxicity that negatively impacts cellular growth and survival .
finally , our work further strengthens the relevance of fission yeast , alongside the more widely accepted budding yeast , to serve as a model organism for studying misfolded proteins linked to human neurodegeneration , opening doors for genetic screens and chemical treatments to dissect the role of genetic factors that regulate -synuclein misfolding and toxicity and contribute to deeper understanding of pd pathogenesis .
cdnas of wild - type ( wt ) and a53 t -synuclein were provided by christopher ross at johns hopkins university .
these -synuclein cdnas were first subcloned into the mammalian expression vector pcdna3.1/c - terminal gfp ( invitrogen ) , fusing -synuclein with gfp at the c - terminus .
the -synuclein - gfp cdnas and gfp cdna were then pcr - amplified and subcloned into the pyes2.1/v5-his - topo budding yeast expression vector ( invitrogen ) or pnmt1/v5-his - topo fission yeast vector ( invitrogen ) .
the -synuclein - gfp fusion yeast vectors were transformed into chemically competent e. coli cells for replication and storage .
the a30p mutant was created using site - directed mutagenesis ( invitrogen ) on wt -synuclein - gfp pyes2.1/v5-his - topo budding yeast expression vector ( invitrogen ) or pnmt1/v5-his - topo as described in sharma et al . and brandis et al .
, respectively [ 45 , 46 ] . the e46k mutant was made similarly created with the following primers : e46k fp : 5atgtaggctccaaaaacaagaagggagtggtgc 3 e46k rp : 5cttggttttggagcctacatagagaacacc 3 
 mutations were sequenced at university of chicago to confirm successful substitution . the parent pyes2.1 vector for budding yeast ( invitrogen ) and parent pnmt1 prep vector ( gift from judy potashkin , rosalind franklin university , ill , usa ) served as controls .
parent budding yeast strains by4741 ( mata his31 leu20 met150 ura30 ) , by4742 ( mathis31 leu20 lys20 met150 ura30 ) , and by4743 ( mata/ his31 leu20 lys20 ura30 ) .
fission yeast strains tcp1 ( h leu1 - 32 ) and sp3 ( h+ leu1 - 32 ) were provided by invitrogen and judy potashkin
( rosalind franklin university of medicine and science , north chicago , ill , usa ) , respectively . 

yeast cells were grown on synthetic - complete media lacking uracil ( sc - ura ) . in the pyes2 vector ,
-synuclein expression was controlled through a tightly regulated galactose - inducible promoter ( gal1 ) .
yeast were grown to mid - log phase in sc - ura glucose ( 2% ) or sc - ura raffinose ( 2% ) media at 30c .
cells were washed with water and diluted to log - phase ( 5  10  cells / ml ) in sc - ura galactose ( 2% ) media to induce -synuclein expression . 

s. pombe strains were transformed with pnmt1 vectors using the lithium - acetate transformation method .
transformed cells were selected by growth on pombe dropout medium - leucine ( pdm - leu ) containing 10  m thiamine .
budding or fission yeast cells at 2.5  10  cells / ml concentration were washed twice with 100  mm nan3 and solubilized in electrophoresis sample buffer ( esb ) .
the esb contained 2% sodium dodecyl sulfate ( sds ) , 80  mm tris ( ph 6.8 ) , 10% glycerol , 1.5% dithiothreitol , 1  mg / ml bromophenol blue , and a cocktail of protease inhibitors and solubilizing agents ( 1% triton - x 100 , 1  mm phenylmethylsulfonyl fluoride , 1  mm benzamidine , 1  mm sodium orthovanadate , 0.7  mg / ml pepstatin a , 0.5  mg / ml leupeptin , 10  mg / ml e64 , 2  mg / ml aprotinin , and 2  mg / ml chymostatin ) .
lysates were electrophoresed at 130 volts on a 10%20% tris - glycine gel ( invitrogen ) with 1x sds running buffer .
gels were transferred to pvdf membranes using a semidry transfer method and probed using the desired antibodies . to detect -synuclein ,
a mouse monoclonal anti - v5-ap antibody ( invitrogen ) was used at 1  :  2000 .
mouse antiphosphoglycerokinase ( pgk ; molecular probes ) was used at 1  :  1000 as a loading control for budding yeast and anti--actin ( abcam ) was used at 1  :  1000 as a loading control for fission yeast . for both
serine-129 phosphorylation blots were probed with a rabbit -synuclein ( phospho s129 ) antibody ( ab59264 ; abcam ) at 1  :  500 , followed by a goat antirabbit secondary antibody ( santa cruz biotechnology ) .
yeast cells were grown in either 10  ml sc - ura+glucose ( budding yeast ) or emm+t ( fission yeast ) overnight at 30c and 200  rpm . to collect cells
they were washed twice with 5  ml h2o , re - suspended in 10  ml h2o , and counted using a hemocytometer to determine cell density .
flasks containing 25  ml sc - ura+galactose ( budding yeast ) or emmt ( fission yeast ) were inoculated to a density of 2.0  10  cells / ml . duplicate spectrophotometer 600  nm absorbance measurements of 1  ml of cells in a plastic cuvette were taken at 0 , 3 , 6 , 12 , 18 , 24 , 36 , and 48 hours after - induction .
a growth curve was generated by plotting the average absorbance readings of three separate experiments versus time in microsoft excel .
yeast cells were grown in either 10  ml sc - ura+glucose ( budding yeast ) or emm+t ( fission yeast ) overnight at 30c and 200  rpm . to collect cells
they were washed twice with 5  ml h2o , resuspended in 10  ml h2o , and counted using a hemocytometer to determine cell density .
cells were diluted 5 fold in a 96 well microtitier plate and spotted onto sc - ura+glucose and sc - ura+galactose ( budding yeast ) or emm+t and emmt ( fission yeast ) growth plates .
cells were grown for 24 hours , and pictures were taken using an hp canoscan scanner .
yeast cells were grown in either 10  ml sc - ura+glucose ( budding yeast ) or emm+t ( fission yeast ) overnight at 30c and 200  rpm . to collect cells
they were washed twice with 5  ml h2o , resuspended in 10  ml h2o , and counted using a hemocytometer to determine cell density .
flasks containing 25  ml sc - ura+galactose ( budding yeast ) or emmt ( fission yeast ) were inoculated to a density of 2.0  10  cells / ml .
1  ml of cell culture was pelleted at 5000  rpm for 1 minute .
the remaining 100  ul of cell culture was vortexed , and 510  ul of sample was pipetted onto a glass slide .
cells were visualized using a nikon te2000-u fluorescent microscope , and images were collected and quantified using metamorph 4.0 software .
all microscopy experiments were conducted twice . for each yeast sample , at each time point ~750 cells of each type were scored for all or some of these localization patterns ( depending on the yeast ) : cytoplasmically diffuse , plasma membrane , aggregate , membrane / diffuse , membrane / aggregate , endomembrane , endomembrane / aggregate , and endomembrane / diffuse ( bottom ) .
yeast cells were grown in either 10  ml sc - ura+glucose ( budding yeast ) or emm+t ( fission yeast ) overnight at 30c and 200  rpm . to collect cells , yeast were pelleted at 1500  g for 5 minutes at 4c .
they were washed three times with 5  ml h2o , resuspended in 10  ml h2o , and counted using a hemocytometer to determine cell density .
flasks containing 25  ml sc - ura+galactose ( budding yeast ) or emmt ( fission yeast ) were inoculated to a density of 2.0  10 cells . at 8 and 18 hours after induction , cell density was determined .
1.8  10 cells were removed and washed once with 1  ml of h2o .
the cells were resuspended in 1  ml of water , and diluted 1  :  1000 .
300  l of 1  :  1000 diluted cells ( 550 cells ) were spread on 150  mm plates containing sc - ura+galactose and sc - ura+glucose ( budding yeast ) or emmt and emm+t ( fission yeast ) , for either two days or three additional days at 30c , and images were taken of plates and analyzed for colony number and size .
 in parkinson 's disease ( pd ) , midbrain dopaminergic neuronal death is linked to the accumulation of aggregated -synuclein . 
 the familial pd mutant form of -synuclein , e46k , has not been thoroughly evaluated yet in an organismal model system . here , we report that e46k resembled wild - type ( wt ) -synuclein in saccharomyces cerevisiae in that it predominantly localized to the plasma membrane , and it did not induce significant toxicity or accumulation . 
 in contrast , in schizosaccharomyces pombe , e46k did not associate with the plasma membrane . instead , in one strain , it extensively aggregated in the cytoplasm and was as toxic as wt . remarkably , in another strain , e46k extensively associated with the endomembrane system and was more toxic than wt . 
 our studies recapitulate and extend aggregation and phospholipid membrane association properties of e46k previously observed in vitro and cell culture . 
 furthermore , it supports the notion that e46k generates toxicity partly due to increased association with endomembrane systems within cells . 
1. Introduction
2. Results
3. Discussion
4. Conclusion
5. Materials and Methods