asymmetric cell division is key to cellular differentiation , and one intriguing aspect of such asymmetry is that a young and immaculate cell can be generated from an aged and deteriorated one ( aguilaniu et  al . , 2003 , higuchi et  al . , 2013 , hughes and gottschling , 2012 , liu et  al . , 2010 , shcheprova et  al . , 2008 ) .
in bacteria , yeast , and specific stem cells , the generation of such rejuvenated progeny includes an asymmetrical inheritance of oxidized and aggregated proteins ( aguilaniu et  al . , 2003 , ogrodnik et  al . , 2014 ,
the retention of protein aggregates in  the yeast progenitor ( mother cell ) requires the protein remodeling factor hsp104 ( erjavec et  al . , 2007 , liu et  al .
2009 ) , the protein deacetylase sir2 ( aguilaniu et  al . , 2003 , orlandi et  al . , 2010 ) , and actin cables together with actin cytoskeletal organizational proteins such as formins , cct , and calmodulin ( liu et  al . , 2010 ,
the control of damage inheritance is dependent also on spatial deposition of damaged / unfolded proteins into specific protein inclusions ( pis ) , such as ipod ( insoluble - protein - deposit ) , junq ( juxta - nuclear - quality - control ) , and inq ( intra - nuclear - quality - control ) ( burri and lithgow , 2004 , kaganovich et  al .
, 2008 , miller et  al . , 2015 , spokoini et  al . , 2012 ) .
formation of such pis are factor - dependent processes , requiring calmodulin and functional actin cables alongside hsp104 ( liu et  al . , 2011 ,
2014 ) , suggesting that the actin cytoskeleton is imperative for both mother cell - biased segregation of aggregates ( liu et  al . , 2011 , song et  al . , 2014 ) and the formation of specific inclusions ( specht et  al . , 2011 ) .
in addition , two members of the hook family of proteins , btn2 and cur1 , control the spatial deposition of misfolded proteins into discrete pis together with chaperones ( e.g. , sis1 ) and small heat shock proteins ( e.g. , hsp42 ) ( malinovska et  al . , 2012 , park et  al . , 2013 , specht et  al . , 2011 ) .
however , the exact mechanisms of how misfolded proteins and aggregates are distinguished and sorted to the different pis and quality - control compartments are not identified ( miller et  al . , 2015 ) .
in addition , the identification of asymmetry - generating genes ( aggs ) controlling inheritance of aggregates and genes required for pi formation has so far been restricted to candidate approaches , suggesting that many components remain to be discovered . in this work ,
we report the results of an unbiased , high - throughput screen for aggs and present a catalog of an organism s complement of genes involved in generating age - asymmetry .
we discovered a previously unknown role for vesicle trafficking , membrane fusion to the vacuole , and vacuole inheritance proteins in the sequestration and fusion of aggregated proteins into cytoprotective ipod sites that are retained in mother cells .
furthermore , we found that the vacuole inheritance adaptor protein vac17 is a limiting member of the aggs and show that this protein regulates asymmetry and replicative lifespan through myo2-dependent effects on endocytosis and spatial protein quality control .
in addition , an unbiased proteomics approach identified members of the endosomal vesicle trafficking process , including the dynamin - like protein vps1 , as physical interactors of the protein disaggregase hsp104 .
we further established that vps1 is involved in aggregate retention and formation of protein inclusions and that vac17 fails to affect these activities in the absence of vps1 , indicating that these two proteins function along the same pathway .
the data highlight that endosomal trafficking to the vacuole is an integral part of a cytoprotective spatial protein quality control affecting the rate of cellular aging .
to embark on unbiased , automatized , screens for aggs , we  generated a mutant library , using synthetic genetic array ( sga ) technology ( see experimental procedures ) , in which the hsp104 gene was replaced by the functional hsp104-gfp fusion .
the hsp104 gene encodes the heat shock protein hsp104 that binds to protein aggregates ( glover and lindquist , 1998 ) , and hsp104-gfp serves as an efficient reporter of such aggregates that can be observed as microscopic intracellular foci and inclusions ( erjavec et  al .
after robot - assisted transfer of cells of the ordered hsp104-gfp - containing arrays to liquid 96-well plates , aggregate formation was elicited by a transient heat shock ( 42c for 30  min ; figure  s1a ) . upon returning to 30c ,
after which no more aggregates were formed , the mother cells were allowed to generate new daughter cells , and the aggregate content in the progenitor and progeny was detected by an online granularity analysis ( molecular devices ) after obtaining images by high - content microscopy ( hcm ) ( figure  s1a ) .
values of the aggregate content in mothers and their daughters were retrieved and mutants ( with deletions in aggs ) deviating from the wild - type value identified . among the 4,600 mutants analyzed , 111 were scored as aggs when a deviation of 25% from the wild - type was used as the cutoff , meaning a 25% increase in daughters inheriting one or more aggregates ( figure  1a ; figure  s1a ; table s1 ) .
the reduction in aggregate asymmetry in the set of mutants identified was not correlated to a general increase ( or decrease ) in aggregation ( figure  s1b ) .
the screen also identified mutants with an enhanced aggregate asymmetry ( table s2 ) , and gene ontology annotation analysis suggests that alterations in the processes related to lipid metabolism and dna repair can boost the mother cells ability to retain aggregates during cytokinesis ( figure  s1c ) .
this set of mutants was not further characterized in this study , which instead focuses on genes ( aggs ) required to generate proper aggregate asymmetry .
aggs were found on all yeast chromosomes but were markedly enriched ( 25% of the hits ) on the right - hand arm of chromosome iii ( figure  s1d ) .
this is interesting considering that duplications of chromosome iii occur reproducibly in yeast cells grown under heat stress and provide the cell with an enhanced tolerance to proteostatic stress ( yona et  al . , 2012 ) .
cross scoring with gene ontology ( go ) , genetic / physical interaction , and keyword databases established that aggs are enriched for actin cytoskeletal functions / binding and intracellular vesicle trafficking processes ( figure  1b ; figures s1e and s1f ) , specifically , genes required for membrane tethering and fusion to the vacuole . such genes included ccz1 ( the guanine nucleotide exchange factor required for membrane tethering and fusion events at the late endosome and vacuole ) ( nordmann et  al . , 2010 ) , the syntaxin - related snares vam3 and syn8 ( mediating docking / fusion of late transport intermediates with the vacuole ) ( burri and lithgow , 2004 ) , and vps8 of the corvet multisubunit tethering complex ( involved in endosomal vesicle tethering and fusion of endosomes to the vacuole ) ( balderhaar and ungermann , 2013 ) ( table s1 ; figures 1b1d ) .
consistently , fab1 mutant cells devoid of  phosphatidylinositol-3,5-bisphosphate ( pi(3,5)p2 ) , a resident signaling lipid on late endosomes , multivesicular bodies ( mvb ) , and vacuoles , required for vesicle fusion to the vacuole ( shaw et  al . , 2003 )
( figures 1c and 1d ) , were also identified in the screen as an agg .
moreover , both vph1 , required for vacuolar  proton pumping as a subunit of the vacuolar - atpase ( v - atpase ) , and rav1 , a member of the rave complex involved in assembly of the v - atpase , were scored as aggs .
these aggs may also be linked to vesicle trafficking as vacuole fusion  has been shown to require vacuolar acidification by the v - atpase ( baars et  al . , 2007 , coonrod et  al . , 2013 ) ( figure  1c ; table s1 ) . that aggs are involved in vesicle and vacuolar fusion 
is noteworthy as misfolded proteins have been shown to become spatially recruited to ipod inclusion sites at the proximity of the vacuole surface ( spokoini et  al . , 2012 )
in addition , we found that the hsp90 chaperone hsp82 acts as an agg and is a central interactor ( physical and genetic ) of several asymmetry - generating processes , including vesicle trafficking , the tor1/skn7 signaling pathway , cytoskeletal organization , chromatin organization / segregation , and lipid signaling ( figure  1c ) . to test whether the quality - control system of protein aggregates  interacts directly with endosomal trafficking systems ( or other systems ) , we performed an unbiased screen for hsp104 interactions during normal growth conditions and heat shock .
hsp104-gfp was immunoprecipitated under mild conditions along with an untagged control both at 30c and 37c .
immunoprecipitation was confirmed by sds - page ( figure  s2a ) , and peptides were identified by liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) .
hsp104 was the most abundant protein present by spectral count as expected ( table s3 ) .
spectral counts in each of the four experiments were compared to the peptideatlas database as described ( miao et  al . , 2013 ,
several components of the vesicle transport system were found to be enriched among hsp104 interactors , including the guanine exchange factor sec7 , the snare disassembly chaperone sec18 , and the vacuole anchor vac8 ( table s3 ) .
analysis of the hsp104 interactions that were seen only during heat shock revealed an even more significant enrichment for proteins involved in vesicle trafficking ( figure  1e ) , suggesting that hsp104 generally associates with the endomembrane system with a more pronounced association during heat shock .
these data indicate that the involvement of membrane trafficking genes in the establishment of damage asymmetry could be linked to an hsp104-mediated interaction between aggregated proteins and components of the vesicle trafficking machinery . among the vacuolar trafficking - related genes identified in the screen
, we found that vac17 and vac8 , required for actin cable - dependent vacuole inheritance ( weisman , 2006 ) , act as aggs ( figure  1c ; table s1 ) .
act1 , myo2 , vac8 , and vac17 encode the four key components of the vacuole inheritance machinery ( figure  2a ) : vac17 serves as an adaptor protein recruiting vacuole vesicles to the actin cable tracks by its dual interaction with vac8 ( on vacuole vesicles ) and the myo2 motor protein ( on actin cables ) ( weisman , 2006 ) .
manual quantification  and complementation of aggregate inheritance defects ( liu et  al . , 2011 , spokoini et  al . , 2012 ) demonstrated that both vac17 ( figure  2b ) and vac8 ( figure  2c ) , similar to act1 and myo2  ( erjavec et  al .
, 2007 , liu et  al . , 2010 , song et  al . ,
2014 ) , are required for mother cell - biased segregation of aggregates . by using a protocol previously described to discriminate between effects on aggregate retention and aggregate removal ( hill et  al . , 2014 ,
2014 ) , we found that vac17 is predominantly affecting aggregate retention ( figure  2d ) .
moreover , while the wild - type allele of vac17 complemented both vacuole  and aggregate inheritance defects when reintroduced into vac17 cells , the vac17bd allele , encoding a vac17 protein unable to interact with myo2 ( tang et  al . , 2003 ) , did not ( figure  2b ) .
consistent with a role for vac17-myo2 interaction for aggregate inheritance , cells containing the myo2-n1304s allele of myo2 , which lacks the vac17-binding domain ( eves et  al . , 2012 ) , displayed a reduced ability to retain protein aggregates in mother cells ( figure  2e ) .
these data indicate that the role of actin cables in establishing aggregate asymmetry ( aguilaniu et  al . , 2003 , erjavec et  al . , 2007 , liu et  al . , 2010
) may be linked to the recruitment of misfolded / aggregated proteins to actin cables by the vac17/8 proteins and myo2 . vac17
and hsp104 aggregates co - localized in about one - third of the cells suggesting that vac17 is , at least to some degree , associated with misfolded / aggregated proteins further strengthening a role for this protein in spatial control of heat - induced aggregates ( figure  2f ) .
however , the absence of  vac17 did not affect inclusion formation or retention of the amyloid , disease - related , huntingtin protein  htt103qp ( dehay and bertolotti , 2006 , wang et  al .
previous studies demonstrated that amyloidic proteins and misfolded proteins formed upon heat shock follow different routes for their deposition ( specht et  al . , 2011 ) ,
and our results suggest that vac17 is only regulating the latter process ( see also data below concerning ubc9 ) .
the vac17 vacuole - adaptor protein is only present at around 20 copies / cell and competes with other adaptor proteins for recruitment of cargo to the myo2 motor protein and actin cables ( eves et  al . , 2012 ) .
to approach whether vac17 might be limiting for aggregate retention in mother cells , we constructed a vac17-overproducing strain by exchanging the weak vac17 promoter with the strong pgpd promoter .
the levels of vac17 were markedly elevated by this promoter exchange ( figure  3a ) , and the retention of both heat - induced and aging - induced aggregates in mother cells were even more pronounced than in wild - type cells ( figures 3b and 3c ) .
this effect was not due to an altered localization of vac17 , as the overproduced protein displayed the same localization pattern as endogenously expressed vac17 : during growth at 30c vac17 is predominantly found at the bud neck or within the bud ( figures s3a and s3b ) , consistent with previous observations ( eves et  al .
, 2012 , jin et  al . , 2009 ) . upon a shift to 38c , however , both endogenously and overexpressed vac17 were relocalized to the mother cell , where 34% and 66% co - localized with hsp104-associated aggregates , respectively ( figures 2f , s3a , and s3b ) .
the localization of the low - abundant vac17 was not the result of background fluorescence or signal bleed - through , as evidenced by a non - gfp control ( figure  s3a ) .
overproduction of vac17 did not affect aggregate inheritance in cells carrying the myo2-n1304s protein unable to bind vac17 suggesting that vac17 acts through its interaction with myo2 also in boosting aggregate asymmetry ( figure  2e ) .
interestingly , inheritance of vacuolar content was not boosted above the level of wild - type cells by overproducing vac17 ( figures 3d and 3e ) , and while overproduction of vac17 completely restored vacuole inheritance in a fab1 mutant it failed to re - establish aggregate  retention ( figure  3f ) . furthermore , while overproduction of vac17 could not limit aggregate inheritance in the myo2-n1304s mutant , vacuole inheritance was modestly improved ( figure  2e ) .
together , these results suggest that vacuolar inheritance defects alone can not explain the defects in aggregate segregation , which therefore may be linked to additional functions of the myo2-vac17 complex ( see below ) .
considering the large number of hits in late endosomal trafficking and membrane fusion , we contemplated the possibility that vac17 might affect aggregate segregation by acting in these  pathways .
consistent with this notion , defects in the hops / corvet ( vps16 : vps16 is a subunit of both hops and corvet ; figure  1d ) multi - subunit tethering complexes required for fusion to the vacuole caused a drastic defect in aggregate retention , and vac17 overproduction failed to affect aggregate inheritance in the absence of these complexes ( figure  3 g ) .
these data , along with the identified requirement for snare proteins ( figures 1c and 1d ; table s1 ) , show that fusion to the vacuole might be required for proper vac17-dependent retention of protein aggregates in mother cells .
however , we can not rule out the possibility that the mutants analyzed suffer from additional defects in vacuole functions that might impact on aggregate inheritance . the aggregate segregation defect in vac17 cells
was accompanied by a reduced ability to deposit misfolded proteins into discrete inclusions ( kaganovich et  al . , 2008 , spokoini et  al . , 2012 ) .
upon a shift from 30c to 38c , the immediate occurrence of multiple stress foci ( sf [ spokoini et  al . , 2012 ] ; also called q - bodies [ escusa - toret et  al . ,
2013 ] , peripheral aggregates [ specht et  al . , 2011 ] , and cytoqs [ miller et  al . , 2015 ] ) and the  subsequent deposition of misfolded / aggregated proteins in  ipod and the juxta - nuclear - quality control compartment ( junq ) can be time resolved ( figure  s3c ) . using hsp104-gfp  together with the sf / ipod / junq - reporter ubc9-rfp , we found that the advancement to the inclusion stage was affected in vac17-deficient cells as these cells displayed multiple hsp104-associated ubc9 sf ( figures 4a4d ) .
in contrast , overproduction of vac17 increased inclusion formation ( figures 4a , 4c , and 4d ) .
as for aggregate inheritance , the effect of vac17 on inclusion formation required its myo2-binding domain ( figure  4b ) .
single - cell analysis demonstrated that vac17 affected the fusion of small sf into inclusions such that the merging of sf was retarded in vac17 cells and accelerated in vac17-overproducing cells ( figure  4d ; figure  s4 ; movies s1 , s2 , and s3 ) . co - staining with fm4 - 64 established that the inclusions of wild - type cells and vac17-overproducing cells often localized to the vicinity of the vacuole ( ipods [ kaganovich et  al . , 2008 ] ) and that the sf of vac17 cells were accompanied by a fragmented vacuole morphology of this mutant ( figures 4e and 4f ; figure  s4 ) . analysis of the small heat shock protein hsp42 , which is specifically localized to sf and ipods ( escusa - toret et  al .
2011 ) , confirmed that vac17 is increasing the efficiency of ipod formation ( figure  s5a ) .
cells lacking the vps16 subunit of the hops / corvet complexes displayed a drastic defect in inclusion formation , and this defect could not , similar to aggregate retention ( figure  3c ) , be suppressed by vac17 overproduction ( figure  5 g ; figure  s5c ; movies s4 and s5 ) .
these data suggest that defects in vacuole  tethering / function inhibit fusion of misfolded proteins / sf into vacuole - proximal ipods in the mother cell , leading to an increased inheritance of damaged proteins by the daughter cell .
cells lacking fab1 , which display defects in vacuole fusion , fission , and tethering and contain one large vacuole rather than fragmented ones ( gary et  al .
, 1998 ) , also display a defect in inclusion formation that was not affected by vac17 overproduction ( figures s5c and s5d ; movies s6 and s7 ) .
this indicates that vacuolar fragmentation is not a prerequisite for a failure to form inclusions .
indeed , salt - stress - induced vacuole fragmentation ( bonangelino et  al . , 2002 , li and kane , 2009 ) demonstrated no significant correlation between the number of vacuoles and the number of heat - induced aggregates ( figures s6a and s6b ) .
similarly , we found no correlation between vacuole numbers and aggregate numbers in aged mother cells ( figure  s6c ) .
the yeast dynamin homolog , vps1 , a resident protein of endosomes required for proper endocytosis to the vacuole ( smaczynska - de rooij et  al . , 2010 ) was identified as an interactor of hsp104 in both untreated and heat - shocked cells ( table s3 ) , and the interaction between vps1 and hsp104 was confirmed by immunoprecipitation ( figure  5a ) .
similar to the trafficking genes identified in the screen , deletion of vps1 resulted in defects in both damage asymmetry and in the formation of protein inclusions , although the effect on inclusion formation was modest ( figures 5b5d ) .
moreover , vac17 failed to affect these processes in cells lacking vps1 ( figures 5b5d ) , suggesting that vps1 and vac17 might act in the same pathway of spatial protein quality control .
vac17 molecules are progressively lost from mother cells during the generation of daughter cells as the protein is transported into the protruding bud and degraded to allow for proper vacuole inheritance ( weisman , 2006 ) .
thus , the vac17 pool needs to be replenished after each cell cycle , and , if not properly compensated for by de novo synthesis , vac17 levels could decline during replicative aging of mother cells .
indeed , we found this to be the case ( figure  6a ) and wondered whether vac17 might become limited in old cells for the management of protein aggregates . in support of this notion ,
overproduction of vac17 reduced the fraction of mother cells displaying aggregates as well as the number of aggregates per aged cell ( figures 6b6d ) , while the opposite result was observed for cells lacking vac17 ( figures 6b6d ) .
elevation of vac17 levels also counteracted age - related vacuolar fragmentation ( figure  6e ) and the decline in endocytotic capacity ( hughes and gottschling , 2012 , tang et  al . , 2008 )
( figure  6f ) , supporting the notion that endocytosis is closely linked to ipod formation and that endocytosis and vesicle fusion to the vacuole is diminished in aged cells .
the data indicate that vac17 possesses a previously unknown role in endocytosis , in addition to its role in vacuole inheritance , and we therefore tested whether cells carrying the myo2 protein ( myo2-n1304s ) , specifically unable to bind vac17 , display reduced endocytosis and found this to be the case both in young and old cells ( figure  6 g ) . to test whether vac17 also affected vacuole - to - vacuole fusion , we analyzed vacuolar fusion during salt - stress recovery and found no evidence for the fusion of vacuoles being retarded in cells lacking vac17 ( figure  s5d ) .
interestingly , improvement of aggregate management and endocytosis by vac17 overproduction was accompanied by a robust extension of lifespan , whereas vac17 deficiency modestly accelerated aging ( figure  7a ) . to evaluate whether the effects of  vac17 acted through its influence on protein quality control ( pqc ) , we tested whether full activity of cytosolic hsp70 chaperone activity and/or the protein disaggregase hsp104 was required for lifespan extension by vac17 .
cytosolic hsp70s were needed for lifespan extension upon vac17 overproduction ( figures 7b and 7c ) , while the hsp104 disaggregase was not ( figure  7c ) , suggesting that pqc functions other than protein disaggregation are required for lifespan control by vac17 .
consistent with the requirement for the hops / corvet complexes for vac17-dependent aggregate fusion ( figure  3 g ) , the vps16 deletion completely blocked lifespan extension by vac17 overproduction ( figure  7d ) . in addition , the myosin - binding domain of vac17 was required for lifespan extension suggesting that interaction with myo2-actin is a prerequisite for vac17-dependent lifespan control , similar to aggregate retention , endocytosis , and ipod formation ( figure  6e ) .
the results demonstrating that vac17 overproduction extend lifespan in a hsp70-dependent manner indicates that vac17-dependent sequestration of misfolded proteins and sf into discrete inclusions ( figures 4a4d ) may be an important process in detoxifying aberrant protein species ( cohen et  al . , 2006 , treusch et  al . , 2009 ) , and we therefore tested whether ubc9 became toxic in vac17 cells , as in cells deficient in cytosolic hsp70 chaperone activity ( ling et  al . , 2014 ) .
we found this to be the case ; lack of vac17 reduced the fitness of cells expressing unfolded ubc9 ( figure  6f ) demonstrating that vac17 is a physiologically relevant aide - de - camp in spatial protein quality control required for mitigating proteotoxicity .
in this paper , using an un - biased imaging approach to discover asymmetry - generating genes ( aggs ) , we found that the adaptor protein vac17 , which is a limiting factor for vacuole inheritance ( weisman , 2006 ) , is a key factor also in ( 1 ) limiting inheritance of protein aggregates , ( 2 ) generating protein inclusions in both heat - stressed and aged cells , and ( 3 ) maintaining endocytosis and vacuole integrity during aging .
specifically , defects in late endocytosis and vesicle fusion to the vacuole lead to defects in protein inclusion formation and subsequent inheritance of protein aggregates , indicating that the aberrant management of damaged proteins observed in aged cells might be linked to a prior decline in endocytosis ( figures 6f and 7 g ) .
mitigating such a decline in vacuole - directed endocytosis by vac17 overproduction counteracted the accumulation of multiple aggregates in aged cells and extended lifespan .
such data are in line with work showing that increased vacuole fusion by osh6 overproduction can retard mother cell aging ( gebre et  al . , 2012 ) and that lifespan extension by caloric restriction requires vesicle - vacuole fusion ( tang et  al . , 2008 ) .
we observed a partial colocalization between vac17 and hsp104-associated aggregates and found that vac17 became increasingly localized to the mother cell during heat stress ( figures 2f and s3 ) .
while such data might indicate that vac17 is affecting inclusion formation and retention by direct interaction with protein aggregates in the mother cell , it is also possible that a fraction of the vac17 pool is aggregating during heat stress .
in fact , a role for vac17 in inclusion formation does not require a direct interaction with protein aggregates as vac17 may be acting upstream the final deposition of misfolded proteins at the surface of the vacuole .
vac17 may be doing so by affecting the rate of vesicle trafficking as discussed , and our data suggest that vac17 is not required for vacuole - vacuole fusions . in addition , we found that vac17 overproduction can suppress severe defects in vacuole inheritance without affecting aggregate inheritance in some mutants analyzed ( figures 2e and 3f ) .
however , although vac17 overexpression can restore vacuole inheritance in some mutants , we can not exclude the possibility that the inherited vacuoles are not fully equivalent , functionally , to wild - type ( anand et  al . , 2009 ) .
therefore , defects in vacuole inheritance may affect aggregate retention not through vacuole inheritance per se , but instead through the generation of daughters and , in turn , mothers with defective vacuoles .
the improvement of endocytosis and extension of lifespan by vac17 overproduction suggest that this protein becomes limiting upon aging , and that the decline in both endocytosis and formation of protein inclusions during aging may be due to a failure to maintain a proper titer of vac17 ( weisman , 2006 ) .
indeed , we found that vac17 levels , normalized to total amount of protein , declined during mother cells aging ( figure  6a ) .
it is possible also that the activity of the endocytic apparatus decline during aging due to an age - related degeneration of actin cytoskeletal dynamics ( gourlay et  al . , 2004 ) and a collapse in vacuolar acidification ( hughes and gottschling , 2012 ) , which is required for  vesicle - vacuole fusion ( coonrod et  al . , 2013 ) .
regardless of  the nature of system failure , it is clear that elevated levels of vac17 alone results in a robust extension of lifespan , pinpointing vac17 as a yeast gerontogene .
we found that vac17 together with the actin - associated protein myo2 is required for the merging of aggregates into ipods , which is interesting since actin , and actin - remodeling proteins such as calmodulin ( cmd ) , is required for heterotypic and homotypic vacuole fusion ( eitzen et  al .
, 2002 ) ( figure  7 g ) , and actin and cmd have previously been shown to be required for proper formation and asymmetrical inheritance of protein inclusions ( liu et  al . , 2010 ,
the concerted effect of vac17-myo2 on endocytosis , vacuolar integrity , and aggregate fusion / ipod formation could be a result of misfolded proteins / stress foci interacting with membrane vesicles ( meriin et  al .
, 2003 ) that could concentrate misfolded proteins at vacuolar - proximal ipod sites with the aid of snare and the hops / corvet tethering complex ( figures 1e and 7 g ) .
indeed , we found that the protein disaggregase hsp104 interacted with several protein components involved in endomembrane trafficking , suggesting that misfolded / aggregated proteins interacting with this disaggregase might hitchhike on trafficking routes leading to the vacuole .
moreover , hsp104-endomembrane protein interactions became more pronounced upon heat shock , indicating that that disaggregation machinery and vesicle trafficking systems are increasingly engaged with each other upon protein aggregation ( figure  1e ) .
the proteins interacting with hsp104 included the dynamin - like protein , vps1 , which appears to act on the same pathway of ipod formation as vac17 .
vps1 is localized on endosomes , and its absence results in a retarded endocytosis to the vacuole and the accumulation of clusters of  endosomes surrounding the vacuole ( smaczynska - de rooij et  al . ,
although the exact mechanisms remain unclear , the vac17-dependent route for aggregate deposition appears to provide the cell with two important features of protein quality control .
first , it ensures that misfolded and potentially cytotoxic proteins are not inherited by the progeny .
some misfolded proteins are imported into the nucleus where their deposition serves as a spatial cytoprotective measure and allows for consecutive degradation by the 26s proteasome ( miller et  al .
it is possible that the same is true for misfolded / aggregated proteins deposited at the vacuolar surface , i.e. , that a proportion of the proteins are translocated into the vacuolar lumen for destruction by vacuolar proteases , a possibility that remains to be elucidated .
the yeast strains used ( s228c background ) were grown in ypd or synthetic drop - out media with corresponding antibiotics .
sga mating to introduce hsp104-gfp into the deletion collection was performed according to published protocols ( costanzo et  al . , 2010 ,
cells were subjected to heat treatment to induce protein aggregation , followed by a recovery period and formaldehyde fixation .
imaging of fixed cells with hsp104-gfp - bound aggregates was performed using a high - content microscope ( imagexpress ) .
acquired images were quantified using metaxpress , and top hits were analyzed for functional enrichment using database for annotation , visualization and integrated discovery ( david ) ( huang et  al .
immunoprecipitations using anti - gfp beads ( chromotek ) were performed on exponentially growing cells expressing hsp104-gfp as well as an untagged control at both 30c and 37c .
eluted proteins were digested with trypsin , peptides were identified by lc - ms / ms , and unweighted spectra were compared to the peptideatlas s.  cerevisiae database to determine enrichment .
protein aggregation was induced at 38c or 42c , followed by live - cell imaging .
single - cell analysis of aggregate fusion was accomplished through 4d confocal imaging of cells attached to concanavalin a ( sigma)-coated microscope plates . for the aggregation of ubc9-rfp ,
expression was induced by growing cells in galactose at 30c and repressed by the addition of glucose before transferring cells to 38c ( kaganovich et  al . , 2008 ) .
all figure data are based on the average of at least three individual experiments , error bars representing sd .
data were tested using an unpaired two - tailed t test , with p values less than 0.05 considered to be significant .
old cells were obtained using the magnetabind biotin - streptavidin approach ( sinclair and guarente , 1997 , smeal et  al . , 1996 ) , consecutively isolating biotin - labeled cells after culturing for 2  days .
replicative lifespan was determined through manual dissection using singer msm micromanipulator ( egilmez et  al . , 1990 ) , and lifespan data were analyzed using a two - tailed mann - whitney u test , with p values less than 0.05 considered as significant .
b.l . designed the agg screen , wrote the journal for image analysis , and , together with x.h . ,
s.m.h . compiled and analyzed the screen data and conducted the follow - up experiments , with the aid of j.g . ,
r.j . p.o.w . performed the biochemical analysis of hsp104 interactions and the follow - up experiments on vps1 .
t.n initiated and coordinated the study , analyzed the data , and , together with b.l . and s.m.h . ,
 summaryage can be reset during mitosis in both yeast and stem cells to generate a young daughter cell from an aged and deteriorated one . 
 this phenomenon requires asymmetry - generating genes ( aggs ) that govern the asymmetrical inheritance of aggregated proteins . using a genome - wide imaging screen to  identify aggs in saccharomyces cerevisiae , we discovered a previously unknown role for endocytosis , vacuole fusion , and the myosin - dependent adaptor protein vac17 in asymmetrical inheritance of misfolded proteins . 
 overproduction of vac17 increases deposition of aggregates into cytoprotective vacuole - associated sites , counteracts age - related breakdown of endocytosis and vacuole integrity , and extends replicative lifespan . 
 the link between damage asymmetry and vesicle trafficking can be explained by a direct interaction between aggregates and vesicles . 
 we also show that the protein disaggregase hsp104 interacts physically with endocytic vesicle - associated proteins , such as the dynamin - like protein , vps1 , which was also shown to be required for vac17-dependent sequestration of protein aggregates . 
 these data demonstrate that two physiognomies of aging  reduced endocytosis and protein aggregation  are interconnected and regulated by vac17 . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions