BACKGROUND
the multisubunit saga  complex is a prototype for transcriptional coactivators that interface between dna binding transcriptional regulators, the chromosomal template and the basal transcriptional machinery  <cit> .
saga is a dynamic entity, with components existing in subcomplexes having related but distinct functions  <cit> .
the ada components of saga  were first identified based on their involvement in regulated transcription in saccharomyces cerevisiae  <cit> .
saga possesses histone acetyltransferase  activity mediated by gcn <dig>  <cit> , which stimulates or represses transcription in a promoter specific fashion  <cit> .
the structural core of saga is composed of a subset of the tbp-associated factors  that are also found in tfiid  <cit> .
spt <dig>  ada <dig> and spt <dig>  members of the tbp-subclass of spt   <cit>  proteins, are located near the core of the complex and are required for its integrity, forming an interface between tafs and other saga-specific components  <cit> .
the additional spt proteins, spt <dig> and spt <dig> are involved in direct interactions with tbp  <cit> .
tra <dig>  a  <dig> kda, phosphatidylinositol-3-kinase related protein  <cit>  is found at the periphery of the complex and interacts with dna-binding transcriptional regulators thus conferring promoter specificity to saga  <cit> .
through mass spectrometry analysis of purified saga complex, other components have been identified  <cit> .
one of these proteins, sus <dig>  is associated with the nuclear pore mrna export machinery  <cit>  suggesting that saga may have a broader role in nuclear processes than previously envisioned.
two forms of spt <dig> have been described.
the full-length protein of  <dig> amino acid residues is found within the saga complex  <cit> .
the second, a c-terminally truncated form likely arising from processing between residues  <dig> and  <dig> is found in the slik  complex  <cit> .
as suggested by the alternate name for this complex, salsa , slik also differs from saga in that it lacks spt <dig>  <cit> .
the absence of spt <dig> can be attributed to the fact that it interacts with spt <dig> in the region deleted in the truncated form  <cit> .
the truncation of spt <dig> and the subsequent loss of spt <dig> may be a mechanism to create a complex that acts without effects mediated through tbp; for example, the derepression of the his <dig> promoter under starvation conditions  <cit> .
a further difference between saga and slik is that rtg <dig>  which is required for the retrograde response pathway in yeast  <cit> , is found specifically in slik  <cit> .
two other rtg proteins, rtg <dig> and rtg <dig> are transcription factors required for the expression of retrograde target genes such as cit <dig>  <cit> .
pray-grant et al.
<cit>  propose that slik facilitates transcriptional activation by rtg <dig> and rtg <dig> and perhaps also at a distinct set of promoters.
however, the exact role of slik is unclear as the interpretation of transcriptional effects upon disruption of rtg <dig> is complicated since rtg <dig> is required for nuclear import of rtg <dig> and rtg <dig>  <cit> .
two models for the relationship between the spt7-containing complexes can be envisioned.
in the first, processing at the c-terminus would occur as part of the biosynthesis of distinct saga and slik complexes.
alternatively, processing might occur during the normal functioning of saga, perhaps to irreversibly signal that a transcriptional event had occurred and/or enabling subsequent activity of the slik complex.
in this study we performed experiments to differentiate between these models.
we show that the conversion of spt <dig> from its full-length saga-form  to its truncated slik-form  and a third c-terminally processed form  occurs in the absence of components of the complex including ada <dig> and spt <dig>  which are required for the integrity of the complex.
processing to these forms occurs rapidly after its synthesis and in the absence of full-length spt <dig>  taken together our results suggest that processing of spt <dig> occurs in the biosynthesis of distinct spt7-containing complexes.
RESULTS
truncated forms of spt <dig> occur in the absence of fully functional saga complex
two forms of spt <dig> have been described that differ in their length  <cit> .
full-length spt <dig> is  <dig> amino acid residues and is found in the saga complex.
this form, spt7saga, migrates with an apparent molecular mass of approximately  <dig> kda on sds-page.
the slik complex contains a c-terminal truncation of spt <dig> that results in an apparent molecular mass of approximately  <dig> kda  <cit> .
based upon the mobility of this form and the differential function of spt <dig> derivatives with c-terminal deletions, spt7slik is predicted to contain a c-terminal truncation of approximately  <dig> amino acids  <cit> .
to detect these forms of spt <dig> we engineered a centromeric plasmid expressing a tap and flag-tagged version of spt <dig> that could be identified after c-terminal truncation .
we first addressed whether this molecule was functional by determining if it would complement a deletion of spt <dig> as assayed by growth on media depleted for inositol.
as shown in figure 1b, a strain lacking spt <dig> is unable to grow on media lacking inositol.
ycpded-tap-flag-spt <dig> allows for near complete restoration of growth.
to determine if the components of the saga or slik complex influence the truncation of spt <dig>  we introduced ycpded-tap-flag-spt <dig> into yeast strain by <dig>  and isogenic strains deleted for gcn <dig>  rtg <dig>  spt <dig>  ubp <dig>  ada <dig> and spt <dig>  cells were grown in ypd media and protein extracts examined for spt <dig> by western blotting with anti-flag antibody .
two proteins with apparent molecular masses of approximately  <dig> and  <dig> kda were present in wild-type cells containing ycpded-tap-flag-spt <dig> but not in control cells .
the size of these molecules suggest that they represent the tap-flag tagged full-length  and slik-forms  of spt <dig>  the ratio of saga:slik forms of spt <dig> was ~3: <dig> in the wild-type strain by <dig> .
this relative amount of spt7slik was not reduced upon disruption of gcn <dig>  rtg <dig>  ada <dig>  spt <dig> or spt <dig>  since spt <dig> and ada <dig> are required for the integrity of the saga complex  <cit> , the presence of spt7slik in these deletion strains indicates that c-terminal processing can occur in the absence of the fully intact and functional complex.
in addition, the wild-type level of spt7slik seen in the spt <dig> deletion background suggests that the spt <dig> interaction with spt <dig> does not alter access of the putative processing enzyme to the cleavage site.
furthermore, the lack of a requirement for rtg <dig> indicates that the slik complex need not fully assemble to allow truncation of spt <dig>
although still apparent, the amount of spt7slik was reduced approximately three-fold in the strain deleted for the ubiquitin protease ubp <dig> .
we note that the ubp8Δ <dig> effect was found to vary from approximately two-fold to five-fold between different experiments .
to determine whether the role of ubp <dig> was the result of its involvement with saga, we examined the spt <dig> forms in a strain deleted for sgf <dig>  a subunit of saga required for the association of ubp <dig> with components of the complex  <cit> .
as shown in figure 2b, deletion of sgf <dig> partially reduced the level of spt7slik.
this effect was not to the same extent as deletion of ubp <dig>  perhaps because low levels of ubp <dig> remain associated with saga in the absence of sgf <dig>  <cit> .
nonetheless, while not eliminating other mechanisms, the result is consistent with a model whereby binding of ubp <dig> to saga may alter the accessibility of the c-terminus of spt <dig> to cleavage.
interestingly, another flag-antibody reactive band, spt7form <dig>  was evident in the sample lanes but not in the control.
since spt7form <dig> was somewhat obscured by a flag antibody cross-reactive band, we analyzed the spt <dig> forms in different strain backgrounds after first purifying tap-flag-spt <dig> on igg-agarose .
after purification a flag-reactive molecule with an apparent mass approximately  <dig> kda less than spt7slik  was revealed.
the observed difference in mass of this molecule relative to intact spt <dig> estimates an end-point at approximately amino acid residue  <dig>  as was the case for spt7slik, the level of this third form of spt <dig> was comparable in each of the deletion strains  with the exception of ubp8Δ <dig> where it was reduced.
c-terminal processing of spt <dig> occurs in the absence of its n-terminal amino acid residues
the finding that spt7slik and spt7form <dig> were formed in the absence of components of saga, including spt <dig> and ada <dig>  suggested that processing is not linked directly to the activity of the complex.
if processing were part of the functional dynamics of saga, that is, occurring as a result of its activities in the regulation of transcription, then one would predict that it would require spt <dig> in a functional form.
we thus examined whether n-terminal deletion derivatives of spt <dig>  expressed in by <dig>  would be processed .
each molecule was n-terminally tap and flag tagged to allow purification and identification of spt <dig> forms.
the two longest derivatives  contain the elements of the protein required for partial function  <cit> .
by comparison, the shortest derivative  lacks the histone fold, which is required for interaction with other components of the complex  <cit> .
a protein reduced by approximately  <dig> kda, an estimated mass consistent with a truncation at the spt7slik processing site, was seen for each of the derivatives .
a further truncation, approximately  <dig> kda smaller than spt7slik and potentially corresponding to processing at the spt7form <dig> site , was evident for spt7581– <dig> and spt7873– <dig> .
note that the third-form equivalent was not seen for spt71037– <dig>  because its putative cleavage site is removed from this construct.
this result suggests that the elements required for processing are intrinsic to the c-terminal end of spt <dig> and that the n-terminus of the protein is not required.
furthermore, the appearance of a cleavage product for spt71037– <dig> suggests that processing can take place independently from saga and is thus not linked to the action of the complex.
for spt7581– <dig>  a fourth form was apparent just below form  <dig> .
we have not characterized this form further and can not exclude that it is a degradation product.
if the forms of spt <dig> were created progressively from one another in response to the activity of the complex, one would predict that the longer forms would be chased into shorter forms.
we analyzed the appearance and disappearance of the spt <dig> forms upon inducing expression of a triple flag-tagged spt <dig> under control of the gal <dig> promoter .
as shown in figure 4a, cells containing gal-flag3-spt <dig> rapidly induce the three spt <dig> forms when shifted from raffinose to galactose containing media but not upon shifting to glucose containing media.
figure 4b is a time course of the appearance and disappearance of the spt <dig> forms after induction in galactose containing media and subsequent shift to media containing glucose.
expression of the forms continued to increase after promoter shut-off until approximately the  <dig> minute time point.
all three forms increased at approximately the same rate.
after  <dig> minutes, the amounts of the spt <dig> forms began to decay.
this was in contrast to total protein, which continued to increase .
similar to their appearance, the three forms disappeared at approximately the same rate.
the longer forms were not chased into the shorter molecules, suggesting that spt7slik and spt7form <dig> are produced early after the synthesis of spt7saga and not sequentially as the result of the action of the complex.
combined with the results above, processing appears inherent to the c-terminus of the molecule and occurs independently from the action of the saga complex.
further characterization of the  <dig> kda form of spt7
to eliminate the possibility that the appearance of spt7form <dig> was related to blotting with flag antibody or to the tap-tag, we expressed n-terminally myc-tagged spt <dig>  separated crude protein extract by sds-page and blotted with anti-myc antibody .
a band approximately  <dig> kda smaller than myc-spt7saga, likely myc-spt7slik was present in wild-type cells containing ycp88-myc-spt <dig> but not in control cells  as was the third-form at an apparent mass of ~ <dig> kda.
to determine if spt7form <dig> was an artifact of the partial tap purification or expression in cells also containing a wild-type copy of spt <dig>  we purified tap-flag-spt <dig> from by <dig>  and by <dig>  through both tandem affinity steps, igg-agarose and calmodulin-sepharose.
as shown in figure 5b, a ~ <dig> kda, flag-detected protein was apparent after purification  and not in a mock purification  in both strain backgrounds.
to examine if spt7form <dig> is found within complexes, tap-flag-spt <dig> was expressed in by <dig> and a crude protein extract applied to a superose  <dig> hr10/ <dig> gel filtration column.
the elution profile of the different forms of spt <dig> was examined by western blotting with anti-flag antibody and the relative amounts of the three forms of spt <dig> determined by densitometry .
all three forms of spt <dig> eluted in fractions corresponding to a high molecular mass, the largest being spt7saga, followed by spt7slik.
spt7form <dig> eluted from the column with an average apparent molecular size slightly less than spt7slik.
to determine if spt7form <dig> was associated with components of the saga complex, extracts were prepared from strains containing n-terminally flag-tagged spt <dig> and tap-tagged versions of gcn <dig>  ada <dig>  ubp <dig>  taf <dig>  spt <dig> and spt <dig>  complexes containing the tap-tagged molecules were partially purified and the presence of the different forms of spt <dig> analyzed by western blotting with anti-flag antibody .
identification of the forms was established by immunoprecipitation of flag-spt <dig> from crude extracts with anti-flag antibody .
as expected from the interaction of spt <dig> with the c-terminal end of spt <dig>  <cit> , spt <dig> interacted only with full-length spt <dig> .
the other saga components interacted with both spt7saga and spt7slik.
spt7form <dig> did not co-purify with the saga components to the extent of either spt7saga or spt7slik.
the function of spt7form <dig> is unknown.
the fact that spt7form <dig> appears as stable as the longer forms is consistent with it having a function, likely distinct from components of the saga complex.
it is interesting to note that spt <dig> has been found to associate with ccl <dig>  rtf <dig> and prp <dig>  and that these interactions are not reciprocated by c-terminally tap-tagged spt <dig> or generally by other components of the saga complex .
in addition, we previously observed a possible saga-independent form of spt <dig> by fractionation of crude extracts on a mono q column  <cit> .
we also must consider an alternative model, whereby spt7form <dig> is an intermediate along a pathway of degradation.
in this regard, we did observe an apparent increase in spt7form <dig> during tap-purification.
CONCLUSIONS
in this report we show that processing of the c-terminus of spt <dig> to the form found in the slik complex and to a novel third form does not require a functional saga complex.
c-terminal truncation occurs in the absence of key components of saga and slik complexes.
spt <dig> and ada <dig> are necessary for the integrity of a complete saga complex with their deletion resulting in severe transcriptional effects  <cit> , yet processing occurs in their absence.
in addition, since the truncated spt <dig> molecules were present in a gcn5Δ <dig> strain, histone acetylation is not required for processing.
also suggesting that processing of spt <dig> is independent of the function of saga, we found that a truncated derivative of spt <dig> that lacks the histone fold is capable of being processed in a strain lacking full-length spt <dig>  finally a time course revealing the appearance and disappearance of the spt <dig> forms showed that spt7saga is not chased into the shorter forms.
we thus suggest that processing of spt <dig> is an independent event in the biosynthesis of distinct spt <dig> containing complexes rather than being linked to transcriptional regulation by the saga complex.
processing of spt <dig> showed partial dependence on the ubiquitin protease ubp <dig>  the role of ubp <dig> is unclear but based on a partial effect upon deletion of sgf <dig>  it may require the association of ubp <dig> with saga.
we were intrigued by the possibility that the effect of ubp <dig> may be related to our previous observation of the ubiquitylation of spt <dig> dependent on the e3-ubiquitin ligase tom <dig>  <cit> ; however, because the relative amounts of the spt <dig> forms appear unchanged in a tom1Δ <dig> strain  we have concluded that these observations are unrelated.
our study has identified a third form of spt <dig> with an apparent molecular mass of ~ <dig> kda.
the identification of this form with three different tagged derivatives suggests that it is not an artifact of tagging; however, we can not fully exclude the possibility that n-terminal tagging results in its appearance.
gel-filtration experiments suggest that spt7form <dig> is found within a complex; albeit, distinct from saga or slik.
as spt <dig> was n-terminally tagged, the mobility of spt7form <dig> on sds-page suggests a molecule containing amino acid residues  <dig> to ~ <dig>  determining the size of the released c-terminal fragment would facilitate mapping of the cleavage site; however, we have been unable to identify this molecule using c-terminally tap-tagged spt <dig>

 the multisubunit saga complex is a prototype for transcriptional coactivators that interface between dna binding transcriptional regulators, the chromosomal template and the basal transcriptional machinery. saga is a dynamic entity, with components existing in subcomplexes having related but distinct functions. the ada components of saga were first identified based on their involvement in regulated transcription in saccharomyces cerevisiae. saga possesses histone acetyltransferase activity mediated by gcn, which stimulates or represses transcription in a promoter specific fashion. the structural core of saga is composed of a subset of the tbp-associated factors that are also found in tfiid. spt ada and spt members of the tbp-subclass of spt proteins, are located near the core of the complex and are required for its integrity, forming an interface between tafs and other saga-specific components. the additional spt proteins, spt and spt are involved in direct interactions with tbp. tra a kda, phosphatidylinositol-3-kinase related protein is found at the periphery of the complex and interacts with dna-binding transcriptional regulators thus conferring promoter specificity to saga. through mass spectrometry analysis of purified saga complex, other components have been identified. one of these proteins, sus is associated with the nuclear pore mrna export machinery suggesting that saga may have a broader role in nuclear processes than previously envisioned. two forms of spt have been described. the full-length protein of amino acid residues is found within the saga complex. the second, a c-terminally truncated form likely arising from processing between residues and is found in the slik complex. as suggested by the alternate name for this complex, salsa, slik also differs from saga in that it lacks spt. the absence of spt can be attributed to the fact that it interacts with spt in the region deleted in the truncated form. the truncation of spt and the subsequent loss of spt may be a mechanism to create a complex that acts without effects mediated through tbp; for example, the derepression of the his promoter under starvation conditions. a further difference between saga and slik is that rtg which is required for the retrograde response pathway in yeast, is found specifically in slik. two other rtg proteins, rtg and rtg are transcription factors required for the expression of retrograde target genes such as cit. pray-grant et al. propose that slik facilitates transcriptional activation by rtg and rtg and perhaps also at a distinct set of promoters. however, the exact role of slik is unclear as the interpretation of transcriptional effects upon disruption of rtg is complicated since rtg is required for nuclear import of rtg and rtg. two models for the relationship between the spt7-containing complexes can be envisioned. in the first, processing at the c-terminus would occur as part of the biosynthesis of distinct saga and slik complexes. alternatively, processing might occur during the normal functioning of saga, perhaps to irreversibly signal that a transcriptional event had occurred and/or enabling subsequent activity of the slik complex. in this study we performed experiments to differentiate between these models. we show that the conversion of spt from its full-length saga-form to its truncated slik-form and a third c-terminally processed form occurs in the absence of components of the complex including ada and spt which are required for the integrity of the complex. processing to these forms occurs rapidly after its synthesis and in the absence of full-length spt taken together our results suggest that processing of spt occurs in the biosynthesis of distinct spt7-containing complexes.  truncated forms of spt occur in the absence of fully functional saga complex two forms of spt have been described that differ in their length. full-length spt is amino acid residues and is found in the saga complex. this form, spt7saga, migrates with an apparent molecular mass of approximately kda on sds-page. the slik complex contains a c-terminal truncation of spt that results in an apparent molecular mass of approximately kda. based upon the mobility of this form and the differential function of spt derivatives with c-terminal deletions, spt7slik is predicted to contain a c-terminal truncation of approximately amino acids. to detect these forms of spt we engineered a centromeric plasmid expressing a tap and flag-tagged version of spt that could be identified after c-terminal truncation. we first addressed whether this molecule was functional by determining if it would complement a deletion of spt as assayed by growth on media depleted for inositol. as shown in figure 1b, a strain lacking spt is unable to grow on media lacking inositol. ycpded-tap-flag-spt allows for near complete restoration of growth. to determine if the components of the saga or slik complex influence the truncation of spt we introduced ycpded-tap-flag-spt into yeast strain by and isogenic strains deleted for gcn rtg spt ubp ada and spt cells were grown in ypd media and protein extracts examined for spt by western blotting with anti-flag antibody. two proteins with apparent molecular masses of approximately and kda were present in wild-type cells containing ycpded-tap-flag-spt but not in control cells. the size of these molecules suggest that they represent the tap-flag tagged full-length and slik-forms of spt the ratio of saga:slik forms of spt was ~3: in the wild-type strain by. this relative amount of spt7slik was not reduced upon disruption of gcn rtg ada spt or spt since spt and ada are required for the integrity of the saga complex, the presence of spt7slik in these deletion strains indicates that c-terminal processing can occur in the absence of the fully intact and functional complex. in addition, the wild-type level of spt7slik seen in the spt deletion background suggests that the spt interaction with spt does not alter access of the putative processing enzyme to the cleavage site. furthermore, the lack of a requirement for rtg indicates that the slik complex need not fully assemble to allow truncation of spt although still apparent, the amount of spt7slik was reduced approximately three-fold in the strain deleted for the ubiquitin protease ubp. we note that the ubp8Δ effect was found to vary from approximately two-fold to five-fold between different experiments. to determine whether the role of ubp was the result of its involvement with saga, we examined the spt forms in a strain deleted for sgf a subunit of saga required for the association of ubp with components of the complex. as shown in figure 2b, deletion of sgf partially reduced the level of spt7slik. this effect was not to the same extent as deletion of ubp perhaps because low levels of ubp remain associated with saga in the absence of sgf. nonetheless, while not eliminating other mechanisms, the result is consistent with a model whereby binding of ubp to saga may alter the accessibility of the c-terminus of spt to cleavage. interestingly, another flag-antibody reactive band, spt7form was evident in the sample lanes but not in the control. since spt7form was somewhat obscured by a flag antibody cross-reactive band, we analyzed the spt forms in different strain backgrounds after first purifying tap-flag-spt on igg-agarose. after purification a flag-reactive molecule with an apparent mass approximately kda less than spt7slik was revealed. the observed difference in mass of this molecule relative to intact spt estimates an end-point at approximately amino acid residue as was the case for spt7slik, the level of this third form of spt was comparable in each of the deletion strains with the exception of ubp8Δ where it was reduced. c-terminal processing of spt occurs in the absence of its n-terminal amino acid residues the finding that spt7slik and spt7form were formed in the absence of components of saga, including spt and ada suggested that processing is not linked directly to the activity of the complex. if processing were part of the functional dynamics of saga, that is, occurring as a result of its activities in the regulation of transcription, then one would predict that it would require spt in a functional form. we thus examined whether n-terminal deletion derivatives of spt expressed in by would be processed. each molecule was n-terminally tap and flag tagged to allow purification and identification of spt forms. the two longest derivatives contain the elements of the protein required for partial function. by comparison, the shortest derivative lacks the histone fold, which is required for interaction with other components of the complex. a protein reduced by approximately kda, an estimated mass consistent with a truncation at the spt7slik processing site, was seen for each of the derivatives. a further truncation, approximately kda smaller than spt7slik and potentially corresponding to processing at the spt7form site, was evident for spt7581– and spt7873–. note that the third-form equivalent was not seen for spt71037– because its putative cleavage site is removed from this construct. this result suggests that the elements required for processing are intrinsic to the c-terminal end of spt and that the n-terminus of the protein is not required. furthermore, the appearance of a cleavage product for spt71037– suggests that processing can take place independently from saga and is thus not linked to the action of the complex. for spt7581– a fourth form was apparent just below form. we have not characterized this form further and can not exclude that it is a degradation product. if the forms of spt were created progressively from one another in response to the activity of the complex, one would predict that the longer forms would be chased into shorter forms. we analyzed the appearance and disappearance of the spt forms upon inducing expression of a triple flag-tagged spt under control of the gal promoter. as shown in figure 4a, cells containing gal-flag3-spt rapidly induce the three spt forms when shifted from raffinose to galactose containing media but not upon shifting to glucose containing media. figure 4b is a time course of the appearance and disappearance of the spt forms after induction in galactose containing media and subsequent shift to media containing glucose. expression of the forms continued to increase after promoter shut-off until approximately the minute time point. all three forms increased at approximately the same rate. after minutes, the amounts of the spt forms began to decay. this was in contrast to total protein, which continued to increase. similar to their appearance, the three forms disappeared at approximately the same rate. the longer forms were not chased into the shorter molecules, suggesting that spt7slik and spt7form are produced early after the synthesis of spt7saga and not sequentially as the result of the action of the complex. combined with the results above, processing appears inherent to the c-terminus of the molecule and occurs independently from the action of the saga complex. further characterization of the kda form of spt7 to eliminate the possibility that the appearance of spt7form was related to blotting with flag antibody or to the tap-tag, we expressed n-terminally myc-tagged spt separated crude protein extract by sds-page and blotted with anti-myc antibody. a band approximately kda smaller than myc-spt7saga, likely myc-spt7slik was present in wild-type cells containing ycp88-myc-spt but not in control cells as was the third-form at an apparent mass of ~ kda. to determine if spt7form was an artifact of the partial tap purification or expression in cells also containing a wild-type copy of spt we purified tap-flag-spt from by and by through both tandem affinity steps, igg-agarose and calmodulin-sepharose. as shown in figure 5b, a ~ kda, flag-detected protein was apparent after purification and not in a mock purification in both strain backgrounds. to examine if spt7form is found within complexes, tap-flag-spt was expressed in by and a crude protein extract applied to a superose hr10/ gel filtration column. the elution profile of the different forms of spt was examined by western blotting with anti-flag antibody and the relative amounts of the three forms of spt determined by densitometry. all three forms of spt eluted in fractions corresponding to a high molecular mass, the largest being spt7saga, followed by spt7slik. spt7form eluted from the column with an average apparent molecular size slightly less than spt7slik. to determine if spt7form was associated with components of the saga complex, extracts were prepared from strains containing n-terminally flag-tagged spt and tap-tagged versions of gcn ada ubp taf spt and spt complexes containing the tap-tagged molecules were partially purified and the presence of the different forms of spt analyzed by western blotting with anti-flag antibody. identification of the forms was established by immunoprecipitation of flag-spt from crude extracts with anti-flag antibody. as expected from the interaction of spt with the c-terminal end of spt, spt interacted only with full-length spt. the other saga components interacted with both spt7saga and spt7slik. spt7form did not co-purify with the saga components to the extent of either spt7saga or spt7slik. the function of spt7form is unknown. the fact that spt7form appears as stable as the longer forms is consistent with it having a function, likely distinct from components of the saga complex. it is interesting to note that spt has been found to associate with ccl rtf and prp and that these interactions are not reciprocated by c-terminally tap-tagged spt or generally by other components of the saga complex. in addition, we previously observed a possible saga-independent form of spt by fractionation of crude extracts on a mono q column. we also must consider an alternative model, whereby spt7form is an intermediate along a pathway of degradation. in this regard, we did observe an apparent increase in spt7form during tap-purification.  in this report we show that processing of the c-terminus of spt to the form found in the slik complex and to a novel third form does not require a functional saga complex. c-terminal truncation occurs in the absence of key components of saga and slik complexes. spt and ada are necessary for the integrity of a complete saga complex with their deletion resulting in severe transcriptional effects, yet processing occurs in their absence. in addition, since the truncated spt molecules were present in a gcn5Δ strain, histone acetylation is not required for processing. also suggesting that processing of spt is independent of the function of saga, we found that a truncated derivative of spt that lacks the histone fold is capable of being processed in a strain lacking full-length spt finally a time course revealing the appearance and disappearance of the spt forms showed that spt7saga is not chased into the shorter forms. we thus suggest that processing of spt is an independent event in the biosynthesis of distinct spt containing complexes rather than being linked to transcriptional regulation by the saga complex. processing of spt showed partial dependence on the ubiquitin protease ubp the role of ubp is unclear but based on a partial effect upon deletion of sgf it may require the association of ubp with saga. we were intrigued by the possibility that the effect of ubp may be related to our previous observation of the ubiquitylation of spt dependent on the e3-ubiquitin ligase tom; however, because the relative amounts of the spt forms appear unchanged in a tom1Δ strain we have concluded that these observations are unrelated. our study has identified a third form of spt with an apparent molecular mass of ~ kda. the identification of this form with three different tagged derivatives suggests that it is not an artifact of tagging; however, we can not fully exclude the possibility that n-terminal tagging results in its appearance. gel-filtration experiments suggest that spt7form is found within a complex; albeit, distinct from saga or slik. as spt was n-terminally tagged, the mobility of spt7form on sds-page suggests a molecule containing amino acid residues to ~ determining the size of the released c-terminal fragment would facilitate mapping of the cleavage site; however, we have been unable to identify this molecule using c-terminally tap-tagged spt 
we conclude that truncation of spt occurs early in the biosynthesis of distinct spt containing complexes rather than being a dynamic process linked to the action of the saga complex in transcriptional regulation. in addition, n-terminally truncated derivatives of spt including a derivative lacking the histone fold, are processed, indicating that the c-terminus of spt is sufficient for processing and that processing does not require functional spt using galactose inducible spt expression, we show that the three forms of spt appear and disappear at approximately the same rate with full-length spt not being chased into spt7slik or spt7form interestingly, reduced levels of spt7slik and spt7form were observed in a strain lacking the saga component ubp suggesting a regulatory role for ubp in the truncation of spt we have found that c-terminal processing of spt to its slik form and to a distinct third form occurs in the absence of the saga complex components gcn spt ada and spt the latter two of which are required for the integrity of the complex.
 spt is an integral component of the multi-subunit saga complex that is required for the expression of ~10% of yeast genes. two forms of spt have been identified, the second of which is truncated at its c-terminus and found in the saga-like complex.  we have found that c-terminal processing of spt to its slik form and to a distinct third form occurs in the absence of the saga complex components gcn spt ada and spt the latter two of which are required for the integrity of the complex. in addition, n-terminally truncated derivatives of spt including a derivative lacking the histone fold, are processed, indicating that the c-terminus of spt is sufficient for processing and that processing does not require functional spt using galactose inducible spt expression, we show that the three forms of spt appear and disappear at approximately the same rate with full-length spt not being chased into spt7slik or spt7form interestingly, reduced levels of spt7slik and spt7form were observed in a strain lacking the saga component ubp suggesting a regulatory role for ubp in the truncation of spt  we conclude that truncation of spt occurs early in the biosynthesis of distinct spt containing complexes rather than being a dynamic process linked to the action of the saga complex in transcriptional regulation. 