BACKGROUND
fungal yapsins constitute a special class of aspartic peptidases characterized by their attachment to membranes through a gpi anchor .
the first and prototypical yapsin member yps <dig>  was discovered in s. cerevisiae because of its ability to process the α-factor precursor c-terminal to lys-arg cleavage sites when overexpressed in a kex2Δ mutant  <cit> .
soon after, it was demonstrated that yps <dig> also recognizes and cleaves c-terminal to single basic residues  in heterogeneous model peptide substrates  <cit> .
typically, several members of the yapsin endopeptidases are expressed per fungal species and they all localize at the cell surface, either associated with the plasma membrane or the cell wall  <cit> .
although their functional significance has yet to be fully elucidated, s. cerevisiae yps <dig> was shown to be involved in the ectodomain shedding of the transmembrane protein msb <dig>  <cit>  and of gpi-anchored proteins including the glucanosyltransferase gas <dig>  the chitin transglycosylase utr <dig> and itself  <cit> .
furthermore, cleavage of gpi-utr <dig> was proposed to constitute a novel cellular response to eliminate excess and/or unfolded gpi-proteins  <cit> .
this hypothesis was recently further substantiated by a high-throughput experiment which revealed that a subset of yeast mutants known to trigger the unfolded protein response  are associated with an increased shedding of both gas <dig> and yps1a.
what controls the activity of yps <dig> and prevents undesirable shedding of important, correctly folded, gpi proteins?
because most aspartic peptidases are initially synthesized as inactive zymogen precursors  <cit> , the activation mechanism of yps <dig> possibly provides an important level of regulation.
another aspect that may control or limit the action of yps <dig> at the cell surface is the nature of the substrate and, more specifically, the context in which the putative cleavage site lies.
for instance, shedding of gas <dig>  yps <dig> itself and utr <dig> is known to occur within s/t-rich domains or clusters that are typically heavily o-glycosylated  <cit> .
could the extent of o-mannosylation in close vicinity of the cleavage site affect its recognition and/or hydrolysis by yps1?
as revealed by previous studies using either a soluble truncated form of yps <dig>  <cit>  or native gpi-anchored yps <dig>  <cit> , the activation mechanism of this endopeptidase is complex and involves multiple proteolytic processing steps.
the pioneer study of cawley et al.
<cit>  demonstrated that a unique feature of yps <dig> is the cleavage of an internal loop located between cys <dig> and cys <dig>  that is absent from the prototypical human pepsin a enzyme .
this cleavage generates a two-subunit endopeptidase , each subunit contributing an asp residue of the catalytic site.
for the native gpi-anchored enzyme, cleavage of this loop was shown to be strictly autocatalytic  <cit> .
furthermore, alternative processing sites were used to generate the two-subunit endopeptidase depending on the extracellular ph suggesting that this processing step occurs once the enzyme has reached the cell surface.
the role of this loop and the significance of its cleavage on yps <dig> function are at present not clear.
an insertion loop at this position is also present in most fungal secreted aspartic peptidases   <cit> , including the yapsins  <cit> , and structures from several saps suggest that it restricts access to the non-prime side of the binding pocket and, accordingly, was given the name ‘n-entrance loop’   <cit> .
assuming that this loop also restricts access to the active site in the case of yps <dig>  and because cleavage of the loop is a late event, we hypothesized that it could be a prerequisite for its shedding activity  <cit> .
however, experimental evidence supporting this hypothesis is lacking.
in the present study, we engineered both a gpi-anchored and a soluble truncated version of yps <dig> where the n-ent loop  is replaced by the short peptide segment found at this location in the yps <dig> homologue yps <dig> .
in addition to assessing directly the role of the n-ent loop in the shedding activity of yps <dig>  by generating a single subunit endopeptidase, this also facilitated the analysis of zymogen activation and its ph dependency.
o-mannosylation of proteins has been described as conferring stability to yeast cell surface proteins  <cit> , and we therefore also addressed whether this modification could interfere with shedding by yps <dig>  in yeast, o-mannosylation on ser and thr residues is catalyzed by a family of o-mannosyltransferases, the pmt enzymes  <cit> .
using yps <dig> and gas <dig> as model gpi substrates, we compared their shedding in the presence or absence of the pmt primarily responsible for their o-mannosylation.
our results clearly demonstrate that pro-peptide removal, even if only partial at ph  <dig> , is essential to generate a functional yps <dig> endopeptidase.
we also show that replacement of the yps <dig> n-ent loop to generate a single chain endopeptidase dramatically increases the release of yps <dig> into the medium.
this suggests that in native yps <dig>  this loop plays a determinant role in regulating its shedding activity.
finally, we observe increased amounts of yps <dig> and gas <dig> into the medium upon disruption of pmt <dig> and pmt <dig>  respectively, the o-mannosyltransferases mainly involved in their o-glycosylation.
it is postulated that hypoglycosylation may play a role in the elimination of misfolded proteins that escaped the er quality control and reached the cell surface.
RESULTS
n-ent loop mutant of gpi-yps <dig> is active in vivo
as cartooned in figure 1a, full length yps <dig>  and its truncated secreted version ssyps <dig>  possess a n-terminal propeptide that is known to be cleaved at the dibasic sites kr <dig> and kr <dig> in ssyps <dig>  <cit> .
while removal of the pro-peptide is suspected to proceed similarly for full length yps <dig>  the exact n-terminus of the mature gpi-anchored endopeptidase has, however, not been determined.
both ssyps <dig> and yps <dig> are also cleaved within an insertion loop to generate an α/β two-subunit endopeptidase  <cit> .
under acidic conditions, the fastest migrating isoform of the β subunit was shown to start at d <dig> suggesting either autocatalytic cleavage c-terminally to n <dig> or cleavage at basic sites upstream  followed by trimming by exoproteolysis as initially suggested by olsen et al.
<cit> .
shedding of native yps <dig> was shown to proceed through autocatalytic cleavage after k <dig> and, as the resulting short peptide  beginning with the sequence 499apgy was secreted, it strongly suggested further proteolytic processing at the only other basic site  prior to the attachment of the gpi anchor at n <dig>  <cit> .figure  <dig>
alignment and structure of scyps <dig> vs casap <dig>  a) schematic representation of native yps <dig> and ssyps <dig> .
cleavage sites known to be used in each forms are indicated by arrows.
b) clustalw alignment  <cit>  of s. cerevisiae yapsins yps <dig> , yps <dig>  and yps <dig>  with secreted aspartyl proteinases sapt <dig> from c. tropicalis , sap <dig> from c. albicans  and sapp <dig> from c. parapsilosis  displaying the insertion loop located between the conserved cysteine residues .
the basic residues  present in the insertion loop of scyps <dig> and scyps <dig> are shown along with the known n-terminus of the fastest migrating form of yps <dig> β subunit .
c) modelled chimeric structure of scyps <dig>  and casap <dig>  obtained from the modweb server.
the n-ent loop  of yps <dig> is shown in green.
the aspartic residues of the catalytic center and the inhibitor pepstatin a are shown in red.
alignment of yps <dig>  with other s. cerevisiae yapsin members  and secreted aspartic peptidases  from c. tropicalis , c. albicans  and c. parapsilosis , revealed that the insertion loop located between a conserved disulfide bridge is significantly larger in scyps <dig> and scyps2p .
as suggested by the chimeric structural model obtained by the modweb server  <cit>  with scyps <dig> and casap <dig> , this loop is superimposed with the so-called n-ent loop of casap <dig>  a second active site flap, which is believed to control access of the substrate to the active site cleft  <cit> .
to investigate the functional role of this n-ent loop in yps <dig> activity, a mutant  where this loop was replaced by the pentapeptide gsvmd found in scyps <dig> was constructed.
overexpression of native yps <dig> was previously shown to partially correct the sds hypersensitivity of kex2Δ mutant yeast  <cit> .
as shown in figure  <dig>  correction of this phenotype was comparable with the native form  and the loop mutant  of yps <dig>  when overexpressed.
in contrast, an active site mutant  of yps1-dl  was as inefficient as the plasmid alone  in alleviating the kex2Δ sds hypersensitivity.
these results therefore indicated that deletion of the n-ent loop is not deleterious for the activity of the endopeptidase.figure  <dig>
both native yps <dig> and yps1-dl correct the sds hypersensitivity of
kex2Δ
yeast.
yps1Δ yeast transformed with prs <dig>  or kex2Δ yps1Δ yeast transformed with the indicated plasmids were first grown overnight in sc medium.
then, serial 1/ <dig> dilutions were spotted onto ypd or ypd supplemented with  <dig> % sds and colonies were grown for 48 h at 30°c.
activation of the n-ent loop mutant requires distinct internal cleavages of the pro-peptide at near neutral  and acidic  conditions
deletion of the n-ent loop is expected to generate a single chain endopeptidase.
to confirm this hypothesis and to analyze the zymogen activation of this mutant form of yps <dig>  a secreted soluble truncated version of yps1-dl  ending with a  <dig> × his-tag at y <dig>  as in ssyps <dig> , was constructed.
the yps1-immunoreactive material secreted from yps1Δ yeast overexpressing ssyps1-dl was then compared to that resulting from the shedding of the yps1Δ yeast overexpressing yps1-dl from  <dig> h cultures in unbuffered  and buffered  sc -ura medium .
at ph  <dig> , two main forms of 48- and  <dig> kda were found in the supernatant of yeast expressing yps1-dl, but some minor species suggestive of partial degradation were also detected below the  <dig> kda marker.
two species were also recovered in the supernatant of yeast expressing ssyps1-dl .
the  <dig> kda species from both ssyps1-dl and yps1-dl were shown to possess an identical n-terminus .
given that the  <dig> × his-tag is c-terminal to y <dig> in ssyps1-dl , this therefore strongly suggests that shedding of the gpi protein yps1-dl occurred through cleavage after k <dig>  as previously determined for the native endopeptidase  <cit> .
at ph  <dig> , a minor form of ~ <dig> kda and a major  <dig> kda form were detected in the medium of yeast overexpressing yps1-dl.
the secreted ~ <dig> kda species suggests that at ph  <dig>  shedding also occurred further downstream from k <dig>  but the precise c-terminal end is not known.
in contrast, three molecular species of ~54-, ~52- and  <dig> kda were immunodetected in the supernatant of yeast expressing ssyps1-dl.
purification by ninta affinity column of these three his-tag species followed by n-terminal sequencing  indicated that the ~ <dig> kda form begins with k <dig> , the ~ <dig> kda form begins with d <dig>  and the ~ <dig> kda species starts with a <dig> .
because a unique sequence was obtained for each of these purified proteins, and at the very n-terminus, this proved that ssyps1-dl produces a single polypeptide chain endopeptidase.figure  <dig>
partial pro-peptide removal is essential for binding of yps1-dl to a pepstatin a-agarose resin.
a) yeast yps1Δ cells transformed with the indicated plasmids were grown at either ph  <dig>  or ph  <dig>  for 24 h and equal amounts of concentrated media  were treated with endo hf and the proteins were separated by 10% sds-page.
after transfer to pvdf membranes, immunoblotting was performed with an yps <dig> antiserum  .
b) yeast yps1Δ cells transformed with ssyps1-dl were grown 24 h at ph  <dig>  and the concentrated supernatant was applied to a pepstatin a-agarose resin as described in “methods”.
aliquots of the starting material , the unbound material  and the eluted fractions  were then separated by 10% sds-page and immunoblotted with an yps <dig> antiserum  .
n-terminal sequencing of purified yps <dig> species
sequenced sample
culture medium ph
molecular mass
identified residues at each cycle*
deduced sequence
*:number in exponent matches the cycle number.
when applied to a pepstatin a-agarose column , the 48 kda band nearly quantitatively absorbed to the affinity column, the 52 kda form bound much less efficiently with the column whereas no binding was detected with the 54 kda species from ssyps1-dl.
since pepstatin a is an aspartic peptidase inhibitor acting as a pseudo substrate, these results indicate that access to the active site cleft of ssyps1-dl requires at least partial removal of the pro-peptide and that its complete removal significantly facilitates substrate/inhibitor binding.
to identify the n-termini of the shed species from yeast expressing yps1-dl, the 48- and 53 kda forms observed at ph  <dig>  were purified by pepstatin a-agarose, separated by sds-page in the presence of dtt, transferred onto a pvdf membrane and each band was subjected to n-terminal sequencing.
the n-terminal of the fastest migrating form  was shown to start with a <dig>  but attempts to sequence the 53 kda band were unsuccessful.
all together, we thus conclude that replacement of the yps <dig> n-ent loop by the short peptide of yps <dig> gives rise to a single chain endopeptidase, as intended, and that depending on the extracellular ph, complete  or only partial  pro-peptide removal is taking place.
although the exact n-terminus of the major shed species at ph  <dig>  could not be determined, its binding to the pepstatin a-agarose column  and its migration  in between the 52 kda and 54 kda species characterized for ssyps1-dl strongly supports that an internal cleavage of the pro-peptide had occurred.
removal of the n-ent loop increases yps <dig> shedding activity
the n-ent loop is proposed to shield the large s <dig> subsite of the binding pocket in fungal aspartic peptidases and to restrict access to the non-prime positions  <cit> .
as known cleavage sites for yps <dig> shedding of gas <dig>  and yps <dig> itself   <cit>  are potentially o-glycosylated at the p <dig> or p <dig> position, respectively, it is conceivable that the n-ent loop partially mask or filter entry of these substrates into the catalytic site thus considerably restricting the efficiency of cleavage under normal conditions.
to evaluate the role of this loop in yps <dig> shedding activity, we compared the release of gas <dig> and either yps <dig> or yps1-dl at both ph  <dig>  and ph  <dig>  .figure  <dig>
yps1-dl has increased shedding activity toward itself and gas <dig> compared to yps <dig>  a) yeast yps1Δ cells transformed with the indicated plasmids were grown 24 h at either ph  <dig>  or ph  <dig> .
equal amounts of the concentrated supernatants, without prior treatment with endo hf, were applied to 10% sds-page and immunoblotted with a gas <dig> antiserum.
b) cell extracts  and concentrated supernatants , treated with endo hf, were prepared from 24 h cultures at either ph  <dig>  or ph  <dig>  of yps1Δ yeast transformed with the indicated plasmids.
after 10% sds-page and transfer to nitrocellulose membranes, proteins were immunodetected with an yps <dig> antiserum .
note that 10-times more material was loaded for the extracellular material compared to cell extracts .
the percentage of total yps1-immunoreactive material  found in cell extracts and supernatants is shown below the western blots.
as previously reported, no gas <dig> was found in the extracellular milieu from yps1Δ yeast expressing the vector alone  at both ph  <dig>  and ph  <dig> .
the overexpression of native yps <dig> or yps1-dl released an 80 kda species of gas <dig> into the medium whereas no gas <dig> immunoreactive forms could be detected upon expression of the active site mutant yps1-dl <dig> .
as estimated by image processing of the bands using imagej , approximately  <dig> -fold  and  <dig> -fold  more gas <dig> was shed by yps1-dl compared to native yps <dig>  since both yps <dig> and yps1-dl are expressed from the same vector, suggesting that similar amounts are being produced, this provided a first indication that deletion of the n-ent loop increases the shedding activity of the endopeptidase.
to confirm that equivalent amounts of endopeptidase are produced by yps <dig> and yps1-dl and to compare their shedding activity toward themselves, the yps <dig> immunoreactive material was analyzed both intra- and extracellularly using the odyssey® fc imaging system to facilitate quantification .
at ph  <dig> , a band of ~ <dig> kda was observed intracellularly and shed species of 53- and  <dig> kda were detected in the supernatant from yps1Δ yeast expressing yps <dig>  nearly 98% of the total yps <dig> immunoreactive material was found intracellularly compared to 2% in the medium.
for yps1Δ yeast expressing yps1-dl, 37% of the yps <dig> 53- and  <dig> kda shed species was found in the medium, an 18-fold increase compared to the shedding of yps <dig>  in yeast expressing the active site mutant of yps1-dl , all yps <dig> immunoreactive material was found in the intracellular  <dig> kda species  and this amounted to approximately 81% of the total  estimated immunoreactive material for yeast expressing yps <dig>  hence, yps <dig> and yps1-dl <dig> are expressed at similar levels.
therefore, the poor recovery of total immunoreactive material  measured for yps-dl  indicates that significant amounts were degraded upon release into the medium as already suggested from the presence of multiple faint species below the  <dig> kda marker in figure  <dig>
a similar pattern was observed at ph  <dig> , except that the  <dig> kda shed species was absent and, in addition to the  <dig> kda species, yps1-dl released a faint band of ~ <dig> kda  that suggests proteolytic processing downstream of k <dig>  most likely c-terminally to kr <dig>  shedding of yps <dig> was less efficient at ph  <dig>  as evidenced by the recovery of 18% of the yps1-immunoreactive material in the medium compared to 37% at ph  <dig>  for yeast expressing yps1-dl.
the total yps1-immunoreactive material associated with yps1-dl and yps1-dl <dig> at ph  <dig>   accounted for 59% and 51%, respectively, to that recovered for native yps <dig>  the lower recovery of intracellular yps1-dl <dig> at ph  <dig>  compared to that measured at ph  <dig>  is obscure, but was observed in two independent experiments.
importantly, the shedding observed with yps1-dl is largely if not exclusively yps1-dependent as no species were released into the medium with the active site mutant yps1-dl <dig> at either ph.
therefore, yeast expressing the n-ent loop mutant produced slightly less amounts of endopeptidase compared to native yps <dig>  particularly at ph  <dig> , but the yps1-dependent shedding was either strongly  or drastically  increased at both ph.
this indicates that the n-ent loop plays a significant role in the yps <dig> shedding activity.
we hypothesized that cleavage of the n-ent loop in native yps <dig>  to generate the two-subunit endopeptidase, could stimulate its activity as observed for the n-ent loop mutant and that it might be intimately linked to its shedding from the cell surface.
if this hypothesis is correct, it predicts that the ratio of immunoreactive material associated with the α subunit over the total yps <dig> immunoreactive material  should be significantly greater in the medium  than inside the cells for native yps <dig>  this was tested under various ph conditions .
as shown in figure  <dig>  the intracellular yps1-immunoreactive material was resolved into a major broad species around 66 kda  and minor species of  <dig>   <dig> and 34 kda; the latter corresponding to the α subunit  <cit> .
the measured intracellular α/t ratios did not exceed  <dig>  across the different ph conditions.
in contrast, the extracellular α/t ratios were significantly greater than the cell associated ratios under acidic conditions , but not at ph  <dig>  .
this therefore supports the hypothesis that the shedding activity of native yps <dig> is stimulated by internal cleavage of the n-ent loop, at least under acidic conditions .figure  <dig>
two-subunit yps <dig> is enriched in the secreted shed species compared to the cell-associated forms.
yeast yps1Δ cells transformed with plasmid yps <dig> was grown 24 h at the indicated ph and aliquots from the cell extract  and concentrated supernatant  were treated with endo hf, separated by 10% sds-page and immunodetected with an yps <dig> antiserum recognizing both the β and α subunits .
the ratio of the immunoreactive yps <dig> α subunit over the total yps <dig> immunoreactive material  is shown at the bottom of each lane.
hypoglycosylation of gas <dig> and yps <dig> in s/t rich domains or clusters stimulates their shedding from the cell surface
as illustrated with yps <dig> and gas <dig> , many potential or known cleavage sites for yps <dig> are located in s/t rich domains or clusters that could be subjected to extensive o-glycosylation by one of the s. cerevisiae pmt enzymes.
addition of mannose residues in close vicinity of the putative cleavage sites could hamper endoproteolysis through steric hindrance .
we thus explored what would be the consequence of preventing or reducing o-glycosylation of these model gpi proteins on their shedding by native yps <dig> and the n-ent loop mutant.
toward this goal, we first investigated which pmt enzyme was responsible for o-glycosylation of native yps <dig> as assessed by gel mobility shift of yps <dig> in cell extracts from yps1Δ, yps1Δ pmt1Δ, yps1Δ pmt2Δ or yps1Δ pmt4Δ mutants.
as shown in figure  <dig>  the absence of pmt <dig> had the most pronounced effect on the major  <dig> kda band that shifted to ~ <dig> kda.
the migration of the truncated version of yps <dig> ending at position y <dig>  was however not altered by the deletion of pmt <dig> .
in contrast, deletion of pmt <dig> modified the migration of the  <dig> kda α subunit.
therefore, the c-terminal s/t rich region of native yps <dig> appears to be modified mostly, if not exclusively, by the pmt <dig> o-mannosyltransferase.figure  <dig>
o
-glycosylation of the yps <dig> s/t-rich domain involves the pmt2
o
-mannosyltransferase.
yeast simple and double mutants transformed with yps <dig> were grown  <dig> h at ph  <dig>  and equal amounts of cell extracts treated with endo hf were analyzed by 10% sds-page and immunoblotted with an yps <dig> antiserum recognizing both the β and α subunits .
only two basic sites, k <dig> and kr <dig>  are present within the yps <dig> c-terminal s/t rich domain  and whether hypoglycosylation of this region affected their use as cleavage sites by native yps <dig> and yps1-dl was tested in a pmt <dig> background.
as shown in figure 7b, compared to that observed in yps1Δ, expression of native yps <dig> in yps1Δ pmt2Δ yeast at ph  <dig>  resulted in a  <dig> -fold increase in the amount of the  <dig> kda shed form  in the medium.
it also led to both an increase  and a shift, resulting from the absence of o-glycosylation, of the ~ <dig> kda species to  <dig> kda .
for yps1-dl, the absence of pmt <dig> had a similar effect on the relative abundance of the  <dig> kda and  <dig> kda species found in the medium .
hence, although shedding of yps1-dl at ph  <dig>  was already significantly more efficient than that observed for native yps <dig> , deletion of pmt <dig> enhanced shedding of both native yps <dig> and yps1-dl.
however, hypoglycosylation of yps <dig>  in addition to stimulate its self-shedding, also led to some release of the catalytically inactive yps1-dl <dig> mutant into the medium .
therefore, hypoglycosylation of the s/t rich region of yps <dig> makes this domain more susceptible to proteolytic processing by both yps <dig> and other peptidases, possibly other yapsin members.
similar results were obtained at ph  <dig> , except that as much as 23% of native yps <dig> was shed from the cell surface in the pmt <dig> background and yps1-dl, under these conditions, was nearly entirely shed from the cell surface .figure  <dig>
hypoglycosylation of yps <dig> stimulates shedding of both native yps <dig> and yps1-dl.
a) schematic view of yps <dig> glycosylation profile and cleavage sites.
potential o-glycosylation sites, as determined by the netoglyc  <dig>  algorithm   <cit> , are indicated with black bars whereas putative and/or known cleavage sites within these regions are illustrated by red bars.
n-glycosylation sites are represented with a symbol.
b) cell extracts  and concentrated supernatants  from  <dig> h cultures at ph  <dig>  of the indicated yeast mutants transformed with plasmid yps <dig> were treated with endo hf, separated by 10% sds-page and immunodetected with an yps <dig> antiserum .
c) cell extracts  and concentrated supernatants , from  <dig> h cultures at ph  <dig> , of the indicated yeast mutants transformed with yps1-dl were treated with endo hf, fractionated by 10% sds-page and immunoblotted with an yps <dig> antiserum .
the percentage of total yps1-immunoreactive material  associated with cell extracts and supernatants is indicated for both yps <dig> and yps1-dl transformed yeast.
d) concentrated supernatants from  <dig> h cultures at ph  <dig>   of the indicated yeast mutants transformed with the catalytically inactive form of yps-dl  and analyzed as in c).
as shown in figure 8a, the potential o-glycosylation sites predicted for gas <dig> are grouped in two regions, the inter-domain separating the glucanosyltransferase domain from the x <dig> domain and the c-terminal s/t rich domain.
in addition to the previously determined cleavage site for yps <dig>  located within the inter-domain  <cit> , several basic sites are present in these two regions.
wild type gas <dig> is known to be o-mannosylated by pmt <dig>  <cit>  and its deletion led to the release of 27% of gas <dig> into the medium compared to 10% in pmt <dig> expressing native yps <dig> .
while the  <dig> kda species was the predominant form accumulating in the medium, several larger species could also be detected  in the pmt <dig> background.
as observed with yps1-dl , hypoglycosylation of gas <dig> also resulted in some yps1-independent shedding .
we thus conclude from these experiments that o-mannosylation is a determinant factor regulating yps1-dependent and -independent shedding of model gpi proteins.figure  <dig>
hypoglycosylation of gas <dig> stimulates shedding by native yps <dig>  a) schematic view of gas1p glycosylation profile and putative cleavage sites as depicted for yps <dig> .
b) cell extracts  and concentrated supernatants  from  <dig> h cultures at ph  <dig>  of the indicated yeast mutants  either transformed  or untransformed with plasmid yps <dig> were separated by 10% sds-page and immunodetected with a gas <dig> antiserum.
asterisks point to faint discrete gas <dig> species detected above the major  <dig> kda species in the extracellular milieu.
the percentage of total gas1-immunoreactive material  associated with cell extracts and supernatants is indicated for all strains.
discussion
most aspartic endopeptidases are initially synthesized as inactive zymogen requiring proteolytic removal of a pro-peptide to become active  <cit> .
using the single-subunit yps <dig> endopeptidase yps1-dl, we showed here that pro-peptide removal is ph dependent.
at ph  <dig> , the gpi-anchored form of yps1-dl was proteolytically processed to generate a mature endopeptidase starting with a <dig>  implying cleavage c-terminal to the kr <dig> basic residues as previously reported for a truncated secreted soluble version of yps1p  <cit> .
although attempts to determine the exact n-termini of yps1-dl at ph  <dig>  were unsuccessful, we demonstrated that the endopeptidase was active at this ph and that only forms missing parts of the pro-peptide could bind to pepstatin a-agarose column.
given the presence of numerous basic residues within the yps <dig> pro-peptide, it is most likely that an intramolecular processing event occurs c-terminal to one of these basic residues at ph  <dig> , possibly k <dig> as originally proposed for the truncated version of the enzyme  <cit> .
yps <dig> substrate specificity is relatively broad, pair basic residues and single basic residues  <cit> .
basic residues are numerous within the s/t rich regions of utr2p, gas1p and yps1p, yet yps1p appears to cleave significantly these substrates only under stressful conditions  <cit> a. we hypothesized that the n-ent loop acts as a filter to restrict hydrolysis of putative cleavage sites present in numerous cell surface proteins.
replacement of the large yps1p n-ent loop between cys <dig> and cys <dig> by the short peptide found at the same location in yps3p was shown here to stimulate its shedding activity either strongly  or dramatically .
the difference in shedding of gas <dig> vs yps1-dl most likely reflects the fact that the endopeptidase  is removed so efficiently from the cell surface that contact between gpi-anchored gas <dig> and yps1-dl is only transient.
importantly, replacement of the loop did not modify the specificity of the enzyme as the same cleavage sites appeared to be used for yps <dig> and yps1-dl.
we earlier proposed that cleavage within the n-ent loop might facilitate the shedding activity of the enzyme  <cit> .
as shown here with the native yps <dig> enzyme,  <dig> to 5-fold more endopeptidase was detected into two-subunits in the medium than inside the cells under acidic conditions , which gives credence to this hypothesis.
the finding that this correlation was not as strong at ph  <dig>  is puzzling.
however, significantly more shedding of a catalytic inactive mutant of native yps <dig>  was observed at ph  <dig>  than at ph  <dig>   <cit> .
this suggests that the lower ratio of two-subunit yps <dig> observed in the medium at ph  <dig>  is due, at least partly, to yps1-independent shedding of single-chain yps <dig>  hence, the n-ent loop indeed seems to restrict access to the active site of the endopeptidase and stabilizes the enzyme at the cell surface.
its deletion or internal cleavage is associated with an increase shedding activity highlighting its regulatory role in yps <dig> function.
it is thus plausible that cleavage of the yps <dig> n-ent loop is stimulated under stressful conditions and further studies should address this important issue.
shedding of yps <dig> substrates typically takes place in s/t rich regions that are predicted to be heavily decorated by o-glycosylation.
in yps <dig>  the o-mannosyltransferase pmt <dig> is preferentially responsible for the addition of mannose residues within the c-terminal s/t rich domain.
for wild type gas <dig>  pmt <dig> is involved but neither pmt <dig> nor pmt <dig>  <cit> .
we showed here that the absence of pmt <dig> stimulates the shedding of both native yps <dig> and the n-ent loop mutant yps1-dl whereas the absence of pmt <dig> increases shedding of gas <dig>  while this increase shedding was largely yps1-dependent, in both cases we provided evidence for the involvement of other endopeptidases as well, possibly other yapsin members.
it therefore suggests that the extent of o-glycosylation is a major determinant for the stability of cell surface proteins by protecting against yps1-dependent and -independent shedding activities.
in line with this hypothesis, it is worth noting that the absence of o-glycosylation leads to aberrant proteolytic processing of the cell surface sensors mid <dig> and the wsc proteins  <cit> .
furthermore, numerous heterologously expressed proteins are subjected to yps <dig> proteolysis  <cit> .
for the heterologous proteins expressed in yeast, the cleavage sites used are not within typical s/t rich regions found in yeast surface proteins and may therefore be hypoglycosylated.
of much interest, differential o-glycosylation at specific sites is now emerging as an important regulator of processing by the mammalian pro-protein convertases  <cit> .
in light of the present study, the function of yeast yapsins may similarly be regulated by differential o-mannosylation resulting, for instance, from stressful conditions.
that hypoglycosylation affects the stability of yeast cell surface proteins through shedding suggests that it may be linked with the elimination of misfolded proteins that escaped the er quality control.
pmt <dig> was recently reported to play an important function in the er-associated degradation  pathway of unfolded proteins  <cit> .
specifically, it was demonstrated that futile cycles of unsuccessful folding ends up with the pmt2-catalyzed o-glycosylation that in turns directs the unfolded protein to the erad pathway.
hence, the absence of pmt <dig>  or an excess of pmt <dig> substrates, likely results in the accumulation of misfolded or unfolded proteins, some escaping erad to reach the cell surface.
indeed, the absence of pmt <dig> was shown to promote post-er degradation of a model misfolded protein, gas1*, which in contrast to native gas <dig> is a substrate for pmt <dig>  <cit> .
deletion of the vacuolar peptidase pep <dig> was found to delay, but not eliminate, degradation of gas1* suggesting that the vacuole may only be one of the alternative mechanisms  <cit> .
in light of the data observed with yps <dig> in the pmt <dig> mutant, it is plausible that shedding at the cell surface by yps1-dependent and yps1-independent processing may as well contribute.
we are currently addressing more directly this hypothesis through proteomic analysis of the secretomes from yeast carrying or not functional pmt and/or yps genes.
CONCLUSIONS
replacement of the n-ent loop of yps <dig> by the short peptide found in the same position in yps <dig>  by producing a single chain endopeptidase, allowed us to elucidate the mechanism by which zymogen activation proceeds at distinct ph for this gpi-anchored endopeptidase.
partial pro-peptide removal is a necessary and critical step to produce a functional enzyme.
without its n-ent loop, the yps <dig> shedding activity is strongly increased suggesting that this domain acts as a major determinant to restrict access to active site and that internal cleavage regulates its activity.
o-glycosylation was also shown to protect against endoproteolysis by native or single-chain yps <dig>  and possibly other yapsin members.
we postulate that misfolded proteins that escape erad might be hypoglycosylated, which would trigger their elimination at the cell surface by yps <dig> and/or other yapsin members.

 fungal yapsins constitute a special class of aspartic peptidases characterized by their attachment to membranes through a gpi anchor. the first and prototypical yapsin member yps was discovered in s. cerevisiae because of its ability to process the α-factor precursor c-terminal to lys-arg cleavage sites when overexpressed in a kex2Δ mutant. soon after, it was demonstrated that yps also recognizes and cleaves c-terminal to single basic residues in heterogeneous model peptide substrates. typically, several members of the yapsin endopeptidases are expressed per fungal species and they all localize at the cell surface, either associated with the plasma membrane or the cell wall. although their functional significance has yet to be fully elucidated, s. cerevisiae yps was shown to be involved in the ectodomain shedding of the transmembrane protein msb and of gpi-anchored proteins including the glucanosyltransferase gas the chitin transglycosylase utr and itself. furthermore, cleavage of gpi-utr was proposed to constitute a novel cellular response to eliminate excess and/or unfolded gpi-proteins. this hypothesis was recently further substantiated by a high-throughput experiment which revealed that a subset of yeast mutants known to trigger the unfolded protein response are associated with an increased shedding of both gas and yps1a. what controls the activity of yps and prevents undesirable shedding of important, correctly folded, gpi proteins? because most aspartic peptidases are initially synthesized as inactive zymogen precursors, the activation mechanism of yps possibly provides an important level of regulation. another aspect that may control or limit the action of yps at the cell surface is the nature of the substrate and, more specifically, the context in which the putative cleavage site lies. for instance, shedding of gas yps itself and utr is known to occur within s/t-rich domains or clusters that are typically heavily o-glycosylated. could the extent of o-mannosylation in close vicinity of the cleavage site affect its recognition and/or hydrolysis by yps1? as revealed by previous studies using either a soluble truncated form of yps or native gpi-anchored yps, the activation mechanism of this endopeptidase is complex and involves multiple proteolytic processing steps. the pioneer study of cawley et al. demonstrated that a unique feature of yps is the cleavage of an internal loop located between cys and cys that is absent from the prototypical human pepsin a enzyme. this cleavage generates a two-subunit endopeptidase, each subunit contributing an asp residue of the catalytic site. for the native gpi-anchored enzyme, cleavage of this loop was shown to be strictly autocatalytic. furthermore, alternative processing sites were used to generate the two-subunit endopeptidase depending on the extracellular ph suggesting that this processing step occurs once the enzyme has reached the cell surface. the role of this loop and the significance of its cleavage on yps function are at present not clear. an insertion loop at this position is also present in most fungal secreted aspartic peptidases, including the yapsins, and structures from several saps suggest that it restricts access to the non-prime side of the binding pocket and, accordingly, was given the name ‘n-entrance loop’. assuming that this loop also restricts access to the active site in the case of yps and because cleavage of the loop is a late event, we hypothesized that it could be a prerequisite for its shedding activity. however, experimental evidence supporting this hypothesis is lacking. in the present study, we engineered both a gpi-anchored and a soluble truncated version of yps where the n-ent loop is replaced by the short peptide segment found at this location in the yps homologue yps. in addition to assessing directly the role of the n-ent loop in the shedding activity of yps by generating a single subunit endopeptidase, this also facilitated the analysis of zymogen activation and its ph dependency. o-mannosylation of proteins has been described as conferring stability to yeast cell surface proteins, and we therefore also addressed whether this modification could interfere with shedding by yps in yeast, o-mannosylation on ser and thr residues is catalyzed by a family of o-mannosyltransferases, the pmt enzymes. using yps and gas as model gpi substrates, we compared their shedding in the presence or absence of the pmt primarily responsible for their o-mannosylation. our results clearly demonstrate that pro-peptide removal, even if only partial at ph , is essential to generate a functional yps endopeptidase. we also show that replacement of the yps n-ent loop to generate a single chain endopeptidase dramatically increases the release of yps into the medium. this suggests that in native yps this loop plays a determinant role in regulating its shedding activity. finally, we observe increased amounts of yps and gas into the medium upon disruption of pmt and pmt respectively, the o-mannosyltransferases mainly involved in their o-glycosylation. it is postulated that hypoglycosylation may play a role in the elimination of misfolded proteins that escaped the er quality control and reached the cell surface.  n-ent loop mutant of gpi-yps is active in vivo as cartooned in figure 1a, full length yps and its truncated secreted version ssyps possess a n-terminal propeptide that is known to be cleaved at the dibasic sites kr and kr in ssyps. while removal of the pro-peptide is suspected to proceed similarly for full length yps the exact n-terminus of the mature gpi-anchored endopeptidase has, however, not been determined. both ssyps and yps are also cleaved within an insertion loop to generate an α/β two-subunit endopeptidase. under acidic conditions, the fastest migrating isoform of the β subunit was shown to start at d suggesting either autocatalytic cleavage c-terminally to n or cleavage at basic sites upstream followed by trimming by exoproteolysis as initially suggested by olsen et al.. shedding of native yps was shown to proceed through autocatalytic cleavage after k and, as the resulting short peptide beginning with the sequence 499apgy was secreted, it strongly suggested further proteolytic processing at the only other basic site prior to the attachment of the gpi anchor at n.figure alignment and structure of scyps vs casap a) schematic representation of native yps and ssyps. cleavage sites known to be used in each forms are indicated by arrows. b) clustalw alignment of s. cerevisiae yapsins yps, yps and yps with secreted aspartyl proteinases sapt from c. tropicalis, sap from c. albicans and sapp from c. parapsilosis displaying the insertion loop located between the conserved cysteine residues. the basic residues present in the insertion loop of scyps and scyps are shown along with the known n-terminus of the fastest migrating form of yps β subunit. c) modelled chimeric structure of scyps and casap obtained from the modweb server. the n-ent loop of yps is shown in green. the aspartic residues of the catalytic center and the inhibitor pepstatin a are shown in red. alignment of yps with other s. cerevisiae yapsin members and secreted aspartic peptidases from c. tropicalis, c. albicans and c. parapsilosis, revealed that the insertion loop located between a conserved disulfide bridge is significantly larger in scyps and scyps2p. as suggested by the chimeric structural model obtained by the modweb server with scyps and casap, this loop is superimposed with the so-called n-ent loop of casap a second active site flap, which is believed to control access of the substrate to the active site cleft. to investigate the functional role of this n-ent loop in yps activity, a mutant where this loop was replaced by the pentapeptide gsvmd found in scyps was constructed. overexpression of native yps was previously shown to partially correct the sds hypersensitivity of kex2Δ mutant yeast. as shown in figure  correction of this phenotype was comparable with the native form and the loop mutant of yps when overexpressed. in contrast, an active site mutant of yps1-dl was as inefficient as the plasmid alone in alleviating the kex2Δ sds hypersensitivity. these results therefore indicated that deletion of the n-ent loop is not deleterious for the activity of the endopeptidase.figure both native yps and yps1-dl correct the sds hypersensitivity of kex2Δ yeast. yps1Δ yeast transformed with prs or kex2Δ yps1Δ yeast transformed with the indicated plasmids were first grown overnight in sc medium. then, serial 1/ dilutions were spotted onto ypd or ypd supplemented with % sds and colonies were grown for 48 h at 30°c. activation of the n-ent loop mutant requires distinct internal cleavages of the pro-peptide at near neutral and acidic conditions deletion of the n-ent loop is expected to generate a single chain endopeptidase. to confirm this hypothesis and to analyze the zymogen activation of this mutant form of yps a secreted soluble truncated version of yps1-dl ending with a × his-tag at y as in ssyps, was constructed. the yps1-immunoreactive material secreted from yps1Δ yeast overexpressing ssyps1-dl was then compared to that resulting from the shedding of the yps1Δ yeast overexpressing yps1-dl from h cultures in unbuffered and buffered sc -ura medium. at ph, two main forms of 48- and kda were found in the supernatant of yeast expressing yps1-dl, but some minor species suggestive of partial degradation were also detected below the kda marker. two species were also recovered in the supernatant of yeast expressing ssyps1-dl. the kda species from both ssyps1-dl and yps1-dl were shown to possess an identical n-terminus. given that the × his-tag is c-terminal to y in ssyps1-dl, this therefore strongly suggests that shedding of the gpi protein yps1-dl occurred through cleavage after k as previously determined for the native endopeptidase. at ph, a minor form of ~ kda and a major kda form were detected in the medium of yeast overexpressing yps1-dl. the secreted ~ kda species suggests that at ph shedding also occurred further downstream from k but the precise c-terminal end is not known. in contrast, three molecular species of ~54-, ~52- and kda were immunodetected in the supernatant of yeast expressing ssyps1-dl. purification by ninta affinity column of these three his-tag species followed by n-terminal sequencing indicated that the ~ kda form begins with k, the ~ kda form begins with d and the ~ kda species starts with a. because a unique sequence was obtained for each of these purified proteins, and at the very n-terminus, this proved that ssyps1-dl produces a single polypeptide chain endopeptidase.figure partial pro-peptide removal is essential for binding of yps1-dl to a pepstatin a-agarose resin. a) yeast yps1Δ cells transformed with the indicated plasmids were grown at either ph  or ph  for 24 h and equal amounts of concentrated media were treated with endo hf and the proteins were separated by 10% sds-page. after transfer to pvdf membranes, immunoblotting was performed with an yps antiserum. b) yeast yps1Δ cells transformed with ssyps1-dl were grown 24 h at ph  and the concentrated supernatant was applied to a pepstatin a-agarose resin as described in “methods”. aliquots of the starting material, the unbound material and the eluted fractions were then separated by 10% sds-page and immunoblotted with an yps antiserum. n-terminal sequencing of purified yps species sequenced sample culture medium ph molecular mass identified residues at each cycle* deduced sequence *:number in exponent matches the cycle number. when applied to a pepstatin a-agarose column, the 48 kda band nearly quantitatively absorbed to the affinity column, the 52 kda form bound much less efficiently with the column whereas no binding was detected with the 54 kda species from ssyps1-dl. since pepstatin a is an aspartic peptidase inhibitor acting as a pseudo substrate, these results indicate that access to the active site cleft of ssyps1-dl requires at least partial removal of the pro-peptide and that its complete removal significantly facilitates substrate/inhibitor binding. to identify the n-termini of the shed species from yeast expressing yps1-dl, the 48- and 53 kda forms observed at ph  were purified by pepstatin a-agarose, separated by sds-page in the presence of dtt, transferred onto a pvdf membrane and each band was subjected to n-terminal sequencing. the n-terminal of the fastest migrating form was shown to start with a but attempts to sequence the 53 kda band were unsuccessful. all together, we thus conclude that replacement of the yps n-ent loop by the short peptide of yps gives rise to a single chain endopeptidase, as intended, and that depending on the extracellular ph, complete or only partial pro-peptide removal is taking place. although the exact n-terminus of the major shed species at ph  could not be determined, its binding to the pepstatin a-agarose column and its migration in between the 52 kda and 54 kda species characterized for ssyps1-dl strongly supports that an internal cleavage of the pro-peptide had occurred. removal of the n-ent loop increases yps shedding activity the n-ent loop is proposed to shield the large s subsite of the binding pocket in fungal aspartic peptidases and to restrict access to the non-prime positions. as known cleavage sites for yps shedding of gas and yps itself are potentially o-glycosylated at the p or p position, respectively, it is conceivable that the n-ent loop partially mask or filter entry of these substrates into the catalytic site thus considerably restricting the efficiency of cleavage under normal conditions. to evaluate the role of this loop in yps shedding activity, we compared the release of gas and either yps or yps1-dl at both ph and ph.figure yps1-dl has increased shedding activity toward itself and gas compared to yps a) yeast yps1Δ cells transformed with the indicated plasmids were grown 24 h at either ph  or ph . equal amounts of the concentrated supernatants, without prior treatment with endo hf, were applied to 10% sds-page and immunoblotted with a gas antiserum. b) cell extracts and concentrated supernatants, treated with endo hf, were prepared from 24 h cultures at either ph  or ph  of yps1Δ yeast transformed with the indicated plasmids. after 10% sds-page and transfer to nitrocellulose membranes, proteins were immunodetected with an yps antiserum. note that 10-times more material was loaded for the extracellular material compared to cell extracts. the percentage of total yps1-immunoreactive material found in cell extracts and supernatants is shown below the western blots. as previously reported, no gas was found in the extracellular milieu from yps1Δ yeast expressing the vector alone at both ph  and ph . the overexpression of native yps or yps1-dl released an 80 kda species of gas into the medium whereas no gas immunoreactive forms could be detected upon expression of the active site mutant yps1-dl. as estimated by image processing of the bands using imagej, approximately -fold and -fold more gas was shed by yps1-dl compared to native yps since both yps and yps1-dl are expressed from the same vector, suggesting that similar amounts are being produced, this provided a first indication that deletion of the n-ent loop increases the shedding activity of the endopeptidase. to confirm that equivalent amounts of endopeptidase are produced by yps and yps1-dl and to compare their shedding activity toward themselves, the yps immunoreactive material was analyzed both intra- and extracellularly using the odyssey® fc imaging system to facilitate quantification. at ph, a band of ~ kda was observed intracellularly and shed species of 53- and kda were detected in the supernatant from yps1Δ yeast expressing yps nearly 98% of the total yps immunoreactive material was found intracellularly compared to 2% in the medium. for yps1Δ yeast expressing yps1-dl, 37% of the yps 53- and kda shed species was found in the medium, an 18-fold increase compared to the shedding of yps in yeast expressing the active site mutant of yps1-dl, all yps immunoreactive material was found in the intracellular kda species and this amounted to approximately 81% of the total estimated immunoreactive material for yeast expressing yps hence, yps and yps1-dl are expressed at similar levels. therefore, the poor recovery of total immunoreactive material measured for yps-dl indicates that significant amounts were degraded upon release into the medium as already suggested from the presence of multiple faint species below the kda marker in figure  a similar pattern was observed at ph, except that the kda shed species was absent and, in addition to the kda species, yps1-dl released a faint band of ~ kda that suggests proteolytic processing downstream of k most likely c-terminally to kr shedding of yps was less efficient at ph as evidenced by the recovery of 18% of the yps1-immunoreactive material in the medium compared to 37% at ph for yeast expressing yps1-dl. the total yps1-immunoreactive material associated with yps1-dl and yps1-dl at ph accounted for 59% and 51%, respectively, to that recovered for native yps the lower recovery of intracellular yps1-dl at ph compared to that measured at ph is obscure, but was observed in two independent experiments. importantly, the shedding observed with yps1-dl is largely if not exclusively yps1-dependent as no species were released into the medium with the active site mutant yps1-dl at either ph. therefore, yeast expressing the n-ent loop mutant produced slightly less amounts of endopeptidase compared to native yps particularly at ph, but the yps1-dependent shedding was either strongly or drastically increased at both ph. this indicates that the n-ent loop plays a significant role in the yps shedding activity. we hypothesized that cleavage of the n-ent loop in native yps to generate the two-subunit endopeptidase, could stimulate its activity as observed for the n-ent loop mutant and that it might be intimately linked to its shedding from the cell surface. if this hypothesis is correct, it predicts that the ratio of immunoreactive material associated with the α subunit over the total yps immunoreactive material should be significantly greater in the medium than inside the cells for native yps this was tested under various ph conditions. as shown in figure  the intracellular yps1-immunoreactive material was resolved into a major broad species around 66 kda and minor species of and 34 kda; the latter corresponding to the α subunit. the measured intracellular α/t ratios did not exceed across the different ph conditions. in contrast, the extracellular α/t ratios were significantly greater than the cell associated ratios under acidic conditions, but not at ph . this therefore supports the hypothesis that the shedding activity of native yps is stimulated by internal cleavage of the n-ent loop, at least under acidic conditions.figure two-subunit yps is enriched in the secreted shed species compared to the cell-associated forms. yeast yps1Δ cells transformed with plasmid yps was grown 24 h at the indicated ph and aliquots from the cell extract and concentrated supernatant were treated with endo hf, separated by 10% sds-page and immunodetected with an yps antiserum recognizing both the β and α subunits. the ratio of the immunoreactive yps α subunit over the total yps immunoreactive material is shown at the bottom of each lane. hypoglycosylation of gas and yps in s/t rich domains or clusters stimulates their shedding from the cell surface as illustrated with yps and gas, many potential or known cleavage sites for yps are located in s/t rich domains or clusters that could be subjected to extensive o-glycosylation by one of the s. cerevisiae pmt enzymes. addition of mannose residues in close vicinity of the putative cleavage sites could hamper endoproteolysis through steric hindrance. we thus explored what would be the consequence of preventing or reducing o-glycosylation of these model gpi proteins on their shedding by native yps and the n-ent loop mutant. toward this goal, we first investigated which pmt enzyme was responsible for o-glycosylation of native yps as assessed by gel mobility shift of yps in cell extracts from yps1Δ, yps1Δ pmt1Δ, yps1Δ pmt2Δ or yps1Δ pmt4Δ mutants. as shown in figure  the absence of pmt had the most pronounced effect on the major kda band that shifted to ~ kda. the migration of the truncated version of yps ending at position y was however not altered by the deletion of pmt. in contrast, deletion of pmt modified the migration of the kda α subunit. therefore, the c-terminal s/t rich region of native yps appears to be modified mostly, if not exclusively, by the pmt o-mannosyltransferase.figure o -glycosylation of the yps s/t-rich domain involves the pmt2 o -mannosyltransferase. yeast simple and double mutants transformed with yps were grown h at ph and equal amounts of cell extracts treated with endo hf were analyzed by 10% sds-page and immunoblotted with an yps antiserum recognizing both the β and α subunits. only two basic sites, k and kr are present within the yps c-terminal s/t rich domain and whether hypoglycosylation of this region affected their use as cleavage sites by native yps and yps1-dl was tested in a pmt background. as shown in figure 7b, compared to that observed in yps1Δ, expression of native yps in yps1Δ pmt2Δ yeast at ph resulted in a -fold increase in the amount of the kda shed form in the medium. it also led to both an increase and a shift, resulting from the absence of o-glycosylation, of the ~ kda species to kda. for yps1-dl, the absence of pmt had a similar effect on the relative abundance of the kda and kda species found in the medium. hence, although shedding of yps1-dl at ph was already significantly more efficient than that observed for native yps, deletion of pmt enhanced shedding of both native yps and yps1-dl. however, hypoglycosylation of yps in addition to stimulate its self-shedding, also led to some release of the catalytically inactive yps1-dl mutant into the medium. therefore, hypoglycosylation of the s/t rich region of yps makes this domain more susceptible to proteolytic processing by both yps and other peptidases, possibly other yapsin members. similar results were obtained at ph, except that as much as 23% of native yps was shed from the cell surface in the pmt background and yps1-dl, under these conditions, was nearly entirely shed from the cell surface.figure hypoglycosylation of yps stimulates shedding of both native yps and yps1-dl. a) schematic view of yps glycosylation profile and cleavage sites. potential o-glycosylation sites, as determined by the netoglyc algorithm, are indicated with black bars whereas putative and/or known cleavage sites within these regions are illustrated by red bars. n-glycosylation sites are represented with a symbol. b) cell extracts and concentrated supernatants from h cultures at ph of the indicated yeast mutants transformed with plasmid yps were treated with endo hf, separated by 10% sds-page and immunodetected with an yps antiserum. c) cell extracts and concentrated supernatants, from h cultures at ph, of the indicated yeast mutants transformed with yps1-dl were treated with endo hf, fractionated by 10% sds-page and immunoblotted with an yps antiserum. the percentage of total yps1-immunoreactive material associated with cell extracts and supernatants is indicated for both yps and yps1-dl transformed yeast. d) concentrated supernatants from h cultures at ph of the indicated yeast mutants transformed with the catalytically inactive form of yps-dl and analyzed as in c). as shown in figure 8a, the potential o-glycosylation sites predicted for gas are grouped in two regions, the inter-domain separating the glucanosyltransferase domain from the x domain and the c-terminal s/t rich domain. in addition to the previously determined cleavage site for yps located within the inter-domain, several basic sites are present in these two regions. wild type gas is known to be o-mannosylated by pmt and its deletion led to the release of 27% of gas into the medium compared to 10% in pmt expressing native yps. while the kda species was the predominant form accumulating in the medium, several larger species could also be detected in the pmt background. as observed with yps1-dl, hypoglycosylation of gas also resulted in some yps1-independent shedding. we thus conclude from these experiments that o-mannosylation is a determinant factor regulating yps1-dependent and -independent shedding of model gpi proteins.figure hypoglycosylation of gas stimulates shedding by native yps a) schematic view of gas1p glycosylation profile and putative cleavage sites as depicted for yps. b) cell extracts and concentrated supernatants from h cultures at ph of the indicated yeast mutants either transformed or untransformed with plasmid yps were separated by 10% sds-page and immunodetected with a gas antiserum. asterisks point to faint discrete gas species detected above the major kda species in the extracellular milieu. the percentage of total gas1-immunoreactive material associated with cell extracts and supernatants is indicated for all strains. discussion most aspartic endopeptidases are initially synthesized as inactive zymogen requiring proteolytic removal of a pro-peptide to become active. using the single-subunit yps endopeptidase yps1-dl, we showed here that pro-peptide removal is ph dependent. at ph , the gpi-anchored form of yps1-dl was proteolytically processed to generate a mature endopeptidase starting with a implying cleavage c-terminal to the kr basic residues as previously reported for a truncated secreted soluble version of yps1p. although attempts to determine the exact n-termini of yps1-dl at ph  were unsuccessful, we demonstrated that the endopeptidase was active at this ph and that only forms missing parts of the pro-peptide could bind to pepstatin a-agarose column. given the presence of numerous basic residues within the yps pro-peptide, it is most likely that an intramolecular processing event occurs c-terminal to one of these basic residues at ph , possibly k as originally proposed for the truncated version of the enzyme. yps substrate specificity is relatively broad, pair basic residues and single basic residues. basic residues are numerous within the s/t rich regions of utr2p, gas1p and yps1p, yet yps1p appears to cleave significantly these substrates only under stressful conditions a. we hypothesized that the n-ent loop acts as a filter to restrict hydrolysis of putative cleavage sites present in numerous cell surface proteins. replacement of the large yps1p n-ent loop between cys and cys by the short peptide found at the same location in yps3p was shown here to stimulate its shedding activity either strongly or dramatically. the difference in shedding of gas vs yps1-dl most likely reflects the fact that the endopeptidase is removed so efficiently from the cell surface that contact between gpi-anchored gas and yps1-dl is only transient. importantly, replacement of the loop did not modify the specificity of the enzyme as the same cleavage sites appeared to be used for yps and yps1-dl. we earlier proposed that cleavage within the n-ent loop might facilitate the shedding activity of the enzyme. as shown here with the native yps enzyme, to 5-fold more endopeptidase was detected into two-subunits in the medium than inside the cells under acidic conditions, which gives credence to this hypothesis. the finding that this correlation was not as strong at ph  is puzzling. however, significantly more shedding of a catalytic inactive mutant of native yps was observed at ph  than at ph . this suggests that the lower ratio of two-subunit yps observed in the medium at ph  is due, at least partly, to yps1-independent shedding of single-chain yps hence, the n-ent loop indeed seems to restrict access to the active site of the endopeptidase and stabilizes the enzyme at the cell surface. its deletion or internal cleavage is associated with an increase shedding activity highlighting its regulatory role in yps function. it is thus plausible that cleavage of the yps n-ent loop is stimulated under stressful conditions and further studies should address this important issue. shedding of yps substrates typically takes place in s/t rich regions that are predicted to be heavily decorated by o-glycosylation. in yps the o-mannosyltransferase pmt is preferentially responsible for the addition of mannose residues within the c-terminal s/t rich domain. for wild type gas pmt is involved but neither pmt nor pmt. we showed here that the absence of pmt stimulates the shedding of both native yps and the n-ent loop mutant yps1-dl whereas the absence of pmt increases shedding of gas while this increase shedding was largely yps1-dependent, in both cases we provided evidence for the involvement of other endopeptidases as well, possibly other yapsin members. it therefore suggests that the extent of o-glycosylation is a major determinant for the stability of cell surface proteins by protecting against yps1-dependent and -independent shedding activities. in line with this hypothesis, it is worth noting that the absence of o-glycosylation leads to aberrant proteolytic processing of the cell surface sensors mid and the wsc proteins. furthermore, numerous heterologously expressed proteins are subjected to yps proteolysis. for the heterologous proteins expressed in yeast, the cleavage sites used are not within typical s/t rich regions found in yeast surface proteins and may therefore be hypoglycosylated. of much interest, differential o-glycosylation at specific sites is now emerging as an important regulator of processing by the mammalian pro-protein convertases. in light of the present study, the function of yeast yapsins may similarly be regulated by differential o-mannosylation resulting, for instance, from stressful conditions. that hypoglycosylation affects the stability of yeast cell surface proteins through shedding suggests that it may be linked with the elimination of misfolded proteins that escaped the er quality control. pmt was recently reported to play an important function in the er-associated degradation pathway of unfolded proteins. specifically, it was demonstrated that futile cycles of unsuccessful folding ends up with the pmt2-catalyzed o-glycosylation that in turns directs the unfolded protein to the erad pathway. hence, the absence of pmt or an excess of pmt substrates, likely results in the accumulation of misfolded or unfolded proteins, some escaping erad to reach the cell surface. indeed, the absence of pmt was shown to promote post-er degradation of a model misfolded protein, gas1*, which in contrast to native gas is a substrate for pmt. deletion of the vacuolar peptidase pep was found to delay, but not eliminate, degradation of gas1* suggesting that the vacuole may only be one of the alternative mechanisms. in light of the data observed with yps in the pmt mutant, it is plausible that shedding at the cell surface by yps1-dependent and yps1-independent processing may as well contribute. we are currently addressing more directly this hypothesis through proteomic analysis of the secretomes from yeast carrying or not functional pmt and/or yps genes.  replacement of the n-ent loop of yps by the short peptide found in the same position in yps by producing a single chain endopeptidase, allowed us to elucidate the mechanism by which zymogen activation proceeds at distinct ph for this gpi-anchored endopeptidase. partial pro-peptide removal is a necessary and critical step to produce a functional enzyme. without its n-ent loop, the yps shedding activity is strongly increased suggesting that this domain acts as a major determinant to restrict access to active site and that internal cleavage regulates its activity. o-glycosylation was also shown to protect against endoproteolysis by native or single-chain yps and possibly other yapsin members. we postulate that misfolded proteins that escape erad might be hypoglycosylated, which would trigger their elimination at the cell surface by yps and/or other yapsin members. 
the yps n-ent loop plays a major role in regulating the shedding activity of the endopeptidase, most likely by limiting access to the active site, and its cleavage in native yps is associated with its shedding. we show here that replacement of the n-entrance loop of yps generates a single chain endopeptidase that undergoes partial or complete pro-peptide removal. the yps1-dependent shedding of two model gpi proteins is also stimulated by the absence of the o-mannosyltransferases, pmt and pmt respectively, involved in o-glycosylation of their ser/thr-rich domains. what regulates its shedding activity at the cell surface is however poorly understood. it is postulated that hypoglycosylation of cell surface proteins, which may occur for misfolded proteins that escaped the er-associated degradation, might target their elimination through shedding by yps and possibly other yapsin members. to investigate the role of this loop on its shedding activity, which typically takes place within ser/thr-rich domains, it was replaced with the short peptide found at the analogous position in yps we also tested whether o-glycosylation might protect against proteolytic processing by yps this glycosylphosphatidylinositol anchored enzyme was recently shown to be involved in the shedding of the gpi proteins utr gas and itself. it was also proposed to be part of a novel quality control mechanism that eliminates excess and/or misfolded gpi proteins. o-glycosylation protects against yps1-dependent and -independent shedding of gpi proteins. partial pro-peptide removal is essential to produce a functional yps endopeptidase. yps is initially synthesized as a zymogen requiring proteolytic activation to remove a pro-peptide and further processing within a large insertion loop generates a two-subunit endopeptidase.
 s. cerevisiae yps is the prototypical aspartic endopeptidase of the fungal yapsin family. this glycosylphosphatidylinositol anchored enzyme was recently shown to be involved in the shedding of the gpi proteins utr gas and itself. it was also proposed to be part of a novel quality control mechanism that eliminates excess and/or misfolded gpi proteins. what regulates its shedding activity at the cell surface is however poorly understood. yps is initially synthesized as a zymogen requiring proteolytic activation to remove a pro-peptide and further processing within a large insertion loop generates a two-subunit endopeptidase. to investigate the role of this loop on its shedding activity, which typically takes place within ser/thr-rich domains, it was replaced with the short peptide found at the analogous position in yps we also tested whether o-glycosylation might protect against proteolytic processing by yps  we show here that replacement of the n-entrance loop of yps generates a single chain endopeptidase that undergoes partial or complete pro-peptide removal. at both ph, the shedding activity of the chimeric endopeptidase toward gas and itself is strongly and drastically increased, respectively. a direct correlation between endoproteolytic cleavage of this loop in native yps and its shedding is observed. the yps1-dependent shedding of two model gpi proteins is also stimulated by the absence of the o-mannosyltransferases, pmt and pmt respectively, involved in o-glycosylation of their ser/thr-rich domains. under these conditions, some yps1-independent shedding is also observed.  partial pro-peptide removal is essential to produce a functional yps endopeptidase. the yps n-ent loop plays a major role in regulating the shedding activity of the endopeptidase, most likely by limiting access to the active site, and its cleavage in native yps is associated with its shedding. o-glycosylation protects against yps1-dependent and -independent shedding of gpi proteins. it is postulated that hypoglycosylation of cell surface proteins, which may occur for misfolded proteins that escaped the er-associated degradation, might target their elimination through shedding by yps and possibly other yapsin members. keywords fungalyapsin/n-entrance loop/zymogen activation/gpi-proteinshedding/post-erquality control/o-glycosylationissue-copyright-statement© the author 2015 