Global Analysis of Protein Sumoylation in Saccharomyces cerevisiae

Abstract

Although the modification of cellular factors by SUMO is an essential process in Saccharomyces cerevisiae, the identities of the substrates remain largely unknown. Using a mass spectrometry-based approach, we have identified 271 new SUMO targets. These substrates play roles in a diverse set of biological processes and greatly expand the scope of SUMO regulation in eukaryotic cells. Transcription appears to be the most prevalent process associated with sumoylation with novel SUMO substrates found in basal transcription machinery for RNA polymerases I, II, and III, pol II transcriptional elongation complexes, and a variety of chromatin remodeling, chromatin modifying, and chromatin silencing complexes. Additionally, our global analysis has revealed a number of interesting biological patterns in the list of SUMO targets including a clustering of sumoylation targets within macromolecular complexes. Although the modification of cellular factors by SUMO is an essential process in Saccharomyces cerevisiae, the identities of the substrates remain largely unknown. Using a mass spectrometry-based approach, we have identified 271 new SUMO targets. These substrates play roles in a diverse set of biological processes and greatly expand the scope of SUMO regulation in eukaryotic cells. Transcription appears to be the most prevalent process associated with sumoylation with novel SUMO substrates found in basal transcription machinery for RNA polymerases I, II, and III, pol II transcriptional elongation complexes, and a variety of chromatin remodeling, chromatin modifying, and chromatin silencing complexes. Additionally, our global analysis has revealed a number of interesting biological patterns in the list of SUMO targets including a clustering of sumoylation targets within macromolecular complexes. Posttranslational modification of proteins is a central mechanism by which biological processes are regulated. One such modification involves the covalent attachment of the small ubiquitin-related polypeptide SUMO 1The abbreviations used are: SUMO, small ubiquitin-like modifier; HA, hemagglutinin; Ni-NTA, nickel-nitrilotriacetic acid; MS, mass spectrometry; TAP, tandem affinity purification.1The abbreviations used are: SUMO, small ubiquitin-like modifier; HA, hemagglutinin; Ni-NTA, nickel-nitrilotriacetic acid; MS, mass spectrometry; TAP, tandem affinity in to cellular substrates SUMO is by a of the the and SUMO including and in of is a covalent the of SUMO to the of in the of a in by a number of including with by attachment to of substrates by SUMO such and in is a central in regulation of by SUMO in and the variety of SUMO targets in to a for sumoylation and in the regulation of biological processes including and is a number of proteins are in Saccharomyces cerevisiae, of substrates have identified to is to be to the of the targets and the a small of a is a set of in the to a of the of SUMO in eukaryotic be the and of a of SUMO targets. we have a global analysis of sumoylation in the a of the and the with a the of and and with the and by with by a the of SUMO for and and the in of to an of by and in and and the proteins in the by the of to a of by in and in by with to and with for in by to the by the of and by mass of by with and of by the and in and by the of and and by tandem mass a mass to III, mass in by by of the most of a of Saccharomyces the III, and the III, for in the the of with for for and for for the used the for of SUMO by of SUMO targets These with a a of to an by and to the in and by for the of the by of and the the by and with of proteins cerevisiae, we a in which the of sumoylation patterns in by of with to sumoylation patterns in the in and in with small in the sumoylation patterns the is to the of the affinity have found sumoylation patterns in sumoylation patterns in which to the of SUMO targets we have found the of a such to SUMO attachment to the SUMO proteins the an a with a of of the affinity with the and affinity by in patterns be found the affinity the of the SUMO with the proteins to the affinity of Using of the SUMO with the of the of targets in our with we to the and proteins III, III, with and and by tandem mass and proteins identified by the with the a the III, proteins identified the of is with the of and of for for and for proteins identified in the of the 271 proteins found to be to the and sumoylation targets. list of of the proteins found in be found in a the list of SUMO substrates appears to be of identified SUMO targets identified in including and and the identified by our of identified SUMO targets the of our and most of the 271 proteins identified in our are to be SUMO the 271 proteins identified in the a of new substrates to be list is and is are proteins are of in is proteins be of and the of in our is the we to and in our of is a to in the our list of SUMO targets. is of which of are have found a to in by be in the in and and identified for in the with and in the has in in is to a in the SUMO and is a SUMO III, the in a sumoylation the of SUMO in the global sumoylation and to an for is the used in for the a to have the proteins identified by a our of are sumoylation targets list of be found in of we identified by and by and III, III, of sumoylation substrates by and are in sumoylation of and and a a a These by by with to the of the and are in of substrates and and and These by by with to the SUMO, in is to of the SUMO substrates identified in our analysis be have in an to the in our have used for a to be a to the affinity is identified in the we the for the with the to a list of of by the of our we a of the sumoylation substrates be by in which targets with a and the of a a to be used a These in and and by with the of the used the of and in the in the SUMO targets are in SUMO of the proteins are in we proteins found to be in our analysis and to sumoylation a to our we in which a to the of and and which identified in our These proteins by and with and found to be sumoylation and sumoylation of our mass and in the of our list of SUMO of SUMO by and analysis of the sumoylation substrates identified in our a number of interesting and patterns be by the of a of the sumoylation targets by biological process which is of the of processes associated with the of SUMO in and roles for sumoylation in a of processes including RNA and a number of sumoylation targets have a in biological interesting for of SUMO substrates by and the of SUMO substrates in a diverse of biological the of SUMO substrates in a of proteins by proteins for the clustering of SUMO substrates we used the the Saccharomyces to for the of for a process in the SUMO list with the in is used to the the of sumoylation to a biological by the of of the proteins to of the biological processes are associated with the sumoylation list with a of are in list of the processes associated with sumoylation the sumoylation substrates to be found in processes by pol of 271 pol II of 271 pol of 271 elongation pol II of 271 chromatin of 271 of 271 of 271 of 271 of 271 of 271 of 271 of 271 in a new interesting with to the regulation of biological processes by Although the sumoylation and the of sumoylation in of transcription including transcription by RNA complexes, transcriptional elongation by RNA II, and of chromatin chromatin modification and is in transcription to sumoylation has the sumoylation of transcription factors and such the of SUMO basal transcription transcriptional elongation complexes, and including and the regulation of transcription by sumoylation is to the of biological processes associated with SUMO, we the of SUMO in a diverse of by used of by to the of of a the number of SUMO substrates found in the is by the most for SUMO a number of targets be found in the and is in with sumoylation is a process with a is sumoylation is to the of of the of the in the of of in in our sumoylation analysis is the clustering of sumoylation targets in macromolecular complexes. Although clustering has for the in in which and are in the and a our we are in and are in which proteins in the of the and factors of the are found to be in our the clustering be to the of associated factors sumoylation within a Although the of clustering is the of in our be a in the regulation of by of identified in in identified in in II identified in in chromatin chromatin identified in in to identified by a chromatin chromatin identified in in silencing to identified by a transcriptional to identified by a to identified by a to identified by a to identified by a to identified by a identified in in to identified by a in a new modification of cellular factors by SUMO is to play a in cellular has to of targets sumoylation is a process and a small of SUMO substrates is a affinity with we have to and in the of 271 proteins are by SUMO in of proteins has associated with sumoylation and SUMO for the in the regulation of of substrates in such a diverse of and processes new the biological of SUMO and the for the analysis of the regulation of new substrates by of our is the the of in SUMO and the SUMO substrates identified in our in in in in our we the sumoylation of proteins in of These proteins be for roles in in and the of the sumoylation of the and and of the and identified in our analysis have identified a number of proteins such and of the play a in the to by in sumoylation our a number of substrates the in in of SUMO and our we of the of sumoylation and the of biological processes and SUMO the of sumoylation in transcription has in such a in our a sumoylation and transcriptional regulation in with the number of new substrates identified roles in transcriptional be the of have identified II the SUMO to be in and cells. the sumoylation of and in in the is in we the sumoylation of and in II, our of an clustering of sumoylation in macromolecular complexes. of of in our clustering an of the regulation of by These a for sumoylation in processes by macromolecular proteins of be the sumoylation of is used a mechanism to the of in a sumoylation to an to a to the by the of the mechanism sumoylation cellular to our have to new SUMO substrates in Although in new targets the number of SUMO targets identified in the number of substrates identified in our of our sumoylation analysis the of to biological patterns and proteins are and a of new substrates to be and the of the sumoylation of factors the biological of Posttranslational modification of proteins is a central mechanism by which biological processes are regulated. One such modification involves the covalent attachment of the small ubiquitin-related polypeptide SUMO 1The abbreviations used are: SUMO, small ubiquitin-like modifier; HA, hemagglutinin; Ni-NTA, nickel-nitrilotriacetic acid; MS, mass spectrometry; TAP, tandem affinity purification.1The abbreviations used are: SUMO, small ubiquitin-like modifier; HA, hemagglutinin; Ni-NTA, nickel-nitrilotriacetic acid; MS, mass spectrometry; TAP, tandem affinity in to cellular substrates SUMO is by a of the the and SUMO including and in of is a covalent the of SUMO to the of in the of a in by a number of including with by attachment to of substrates by SUMO such and in is a central in regulation of by SUMO in and the variety of SUMO targets in to a for sumoylation and in the regulation of biological processes including and is a number of proteins are in Saccharomyces cerevisiae, of substrates have identified to is to be to the of the targets and the a small of a is a set of in the to a of the of SUMO in eukaryotic be the and of a of SUMO targets. we have a global analysis of sumoylation in the a of the and the with a the of and and with the and by with by a the of SUMO for and and the in of to an of by and in and and the proteins in the by the of to a of by in and in by with to and with for in by to the by the of and by mass of by with and of by the and in and by the of and and by tandem mass a mass to III, mass in by by of the most of a of Saccharomyces the III, and the III, for in the the of with for for and for for the used the for of SUMO by of SUMO targets These with a a of to an by and to the in and by for the of the by of and the the by and with the a of the and the with a the of and and with the and by with by a the of SUMO for and and the in of to an of by and in and and the proteins in the by the of to a of by in and in by with to and with for in by to the by the of and by mass of by with and of by the and in and by the of and and by tandem mass a mass to III, mass in by by of the most of a of Saccharomyces the III, and the III, for in the the of with for for and for for the used the for of SUMO by of SUMO targets These with a a of to an by and to the in and by for the of the by of and the the by and with of proteins cerevisiae, we a in which the of sumoylation patterns in by of with to sumoylation patterns in the in and in with small in the sumoylation patterns the is to the of the affinity have found sumoylation patterns in sumoylation patterns in which to the of SUMO targets we have found the of a such to SUMO attachment to the SUMO proteins the an a with a of of the affinity with the and affinity by in patterns be found the affinity the of the SUMO with the proteins to the affinity of Using of the SUMO with the of the of targets in our with we to the and proteins III, III, with and and by tandem mass and proteins identified by the with the a the III, proteins identified the of is with the of and of for for and for proteins identified in the of the 271 proteins found to be to the and sumoylation targets. list of of the proteins found in be found in a the list of SUMO substrates appears to be of identified SUMO targets identified in including and and the identified by our of identified SUMO targets the of our and most of the 271 proteins identified in our are to be SUMO the 271 proteins identified in the a of new substrates to be list is and is are proteins are of in is proteins be of and the of in our is the we to and in our of is a to in the our list of SUMO targets. is of which of are have found a to in by be in the in and and identified for in the with and in the has in in is to a in the SUMO and is a SUMO III, the in a sumoylation the of SUMO in the global sumoylation and to an for is the used in for the a to have the proteins identified by a our of are sumoylation targets list of be found in of we identified by and by and III, III, in is to of the SUMO substrates identified in our analysis be have in an to the in our have used for a to be a to the affinity is identified in the we the for the with the to a list of of by the of our we a of the sumoylation substrates be by in which targets with a and the of a a to be used a These in and and by with the of the used the of and in the in the SUMO targets are in SUMO of the proteins are in we proteins found to be in our analysis and to sumoylation a to our we in which a to the of and and which identified in our These proteins by and with and found to be sumoylation and sumoylation of our mass and in the of our list of SUMO of SUMO by and analysis of the sumoylation substrates identified in our a number of interesting and patterns be by the of a of the sumoylation targets by biological process which is of the of processes associated with the of SUMO in and roles for sumoylation in a of processes including RNA and a number of sumoylation targets have a in biological interesting for of SUMO substrates by and the of SUMO substrates in a diverse of biological the of SUMO substrates in a of proteins by proteins for the clustering of SUMO substrates we used the the Saccharomyces to for the of for a process in the SUMO list with the in is used to the the of sumoylation to a biological by the of of the proteins to of the biological processes are associated with the sumoylation list with a of are in list of the processes associated with sumoylation the sumoylation substrates to be found in processes by pol of 271 pol II of 271 pol of 271 elongation pol II of 271 chromatin of 271 of 271 of 271 of 271 of 271 of 271 of 271 of 271 in a new interesting with to the regulation of biological processes by Although the sumoylation and the of sumoylation in of transcription including transcription by RNA complexes, transcriptional elongation by RNA II, and of chromatin chromatin modification and is in transcription to sumoylation has the sumoylation of transcription factors and such the of SUMO basal transcription transcriptional elongation complexes, and including and the regulation of transcription by sumoylation is to the of biological processes associated with SUMO, we the of SUMO in a diverse of by used of by to the of of a the number of SUMO substrates found in the is by the most for SUMO a number of targets be found in the and is in with sumoylation is a process with a is sumoylation is to the of of the of the in the of of in in our sumoylation analysis is the clustering of sumoylation targets in macromolecular complexes. Although clustering has for the in in which and are in the and a our we are in and are in which proteins in the of the and factors of the are found to be in our the clustering be to the of associated factors sumoylation within a Although the of clustering is the of in our be a in the regulation of by of identified in in identified in in II identified in in chromatin chromatin identified in in to identified by a chromatin chromatin identified in in silencing to identified by a transcriptional to identified by a to identified by a to identified by a to identified by a to identified by a identified in in to identified by a in a new of proteins cerevisiae, we a in which the of sumoylation patterns in by of with to sumoylation patterns in the in and in with small in the sumoylation patterns the is to the of the affinity have found sumoylation patterns in sumoylation patterns in which to the of SUMO targets we have found the of a such to SUMO attachment to the SUMO proteins the an a with a of of the affinity with the and affinity by in patterns be found the affinity the of the SUMO with the proteins to the affinity of Using of the SUMO with the of the of targets in our with we to the and proteins III, III, with and and by tandem mass and proteins identified by the with the a the III, proteins identified the of is with the of and of for for and for proteins identified in the of the 271 proteins found to be to the and sumoylation targets. list of of the proteins found in be found in a the list of SUMO substrates appears to be of identified SUMO targets identified in including and and the identified by our of identified SUMO targets the of our and most of the 271 proteins identified in our are to be SUMO Although the 271 proteins identified in the a of new substrates to be list is and is are proteins are of in is proteins be of and the of in our is the we to and in our of is a to in the our list of SUMO targets. is of which of are have found a to in by be in the in and and identified for in the with and in the has in in is to a in the SUMO and is a SUMO III, the in a sumoylation the of SUMO in the global sumoylation and to an for is the used in for the a to have the proteins identified by a our of are sumoylation targets list of be found in of we identified by and by and III, III, in is to of the SUMO substrates identified in our analysis be have in an to the in our have used for a to be a to the affinity is identified in the we the for the with the to a list of of by the of our we a of the sumoylation substrates be by in which targets with a and the of a a to be used a These in and and by with the of the used the of and in the in the SUMO targets are in SUMO of the proteins are in we proteins found to be in our analysis and to sumoylation a to our we in which a to the of and and which identified in our These proteins by and with and found to be sumoylation and sumoylation of our mass and in the of our list of SUMO of SUMO by and analysis of the sumoylation substrates identified in our a number of interesting and patterns be by the of a of the sumoylation targets by biological process which is of the of processes associated with the of SUMO in and roles for sumoylation in a of processes including RNA and a number of sumoylation targets have a in biological interesting for proteins by proteins for the clustering of SUMO substrates we used the the Saccharomyces to for the of for a process in the SUMO list with the in is used to the the of sumoylation to a biological by the of of the proteins to of the biological processes are associated with the sumoylation list with a of are in list of the processes associated with sumoylation the sumoylation substrates to be found in interesting with to the regulation of biological processes by Although the sumoylation and the of sumoylation in of transcription including transcription by RNA complexes, transcriptional elongation by RNA II, and of chromatin chromatin modification and is in transcription to sumoylation has the sumoylation of transcription factors and such the of SUMO basal transcription transcriptional elongation complexes, and including and the regulation of transcription by sumoylation is to the of biological processes associated with SUMO, we the of SUMO in a diverse of by used of by to the of of a the number of SUMO substrates found in the is by the most for SUMO a number of targets be found in the and is in with sumoylation is a process with a is sumoylation is to the of of the of the in the of of in in our sumoylation analysis is the clustering of sumoylation targets in macromolecular complexes. Although clustering has for the in in which and are in the and a our we are in and are in which proteins in the of the and factors of the are found to be in our the clustering be to the of associated factors sumoylation within a Although the of clustering is the of in our be a in the regulation of by modification of cellular factors by SUMO is to play a in cellular has to of targets sumoylation is a process and a small of SUMO substrates is a affinity with we have to and in the of 271 proteins are by SUMO in of proteins has associated with sumoylation and SUMO for the in the regulation of of substrates in such a diverse of and processes new the biological of SUMO and the for the analysis of the regulation of new substrates by of our is the the of in SUMO and the SUMO substrates identified in our in in in in our we the sumoylation of proteins in of These proteins be for roles in in and the of the sumoylation of the and and of the and identified in our analysis have identified a number of proteins such and of the play a in the to by in sumoylation our a number of substrates the in in of SUMO and our we of the of sumoylation and the of biological processes and SUMO the of sumoylation in transcription has in such a in our a sumoylation and transcriptional regulation in with the number of new substrates identified roles in transcriptional be the of have identified II the SUMO to be in and cells. the sumoylation of and in in the is in we the sumoylation of and in II, our of an clustering of sumoylation in macromolecular complexes. of of in our clustering an of the regulation of by These a for sumoylation in processes by macromolecular proteins of be the sumoylation of is used a mechanism to the of in a sumoylation to an to a to the by the of the mechanism sumoylation cellular to our have to new SUMO substrates in Although in new targets the number of SUMO targets identified in the number of substrates identified in our of our sumoylation analysis the of to biological patterns and proteins are and a of new substrates to be and the of the sumoylation of factors the biological of modification of cellular factors by SUMO is to play a in cellular has to of targets sumoylation is a process and a small of SUMO substrates is a affinity with we have to and in the of 271 proteins are by SUMO in of proteins has associated with sumoylation and SUMO for the in the regulation of of substrates in such a diverse of and processes new the biological of SUMO and the for the analysis of the regulation of new substrates by of our is the the of in SUMO and the SUMO substrates identified in our in in in in our we the sumoylation of proteins in of These proteins be for roles in in and the of the sumoylation of the and and of the and identified in our analysis have identified a number of proteins such and of the play a in the to by in sumoylation our a number of substrates the in in of SUMO and our we of the of sumoylation and the of biological processes and SUMO the of sumoylation in transcription has in such a in our a sumoylation and transcriptional regulation in with the number of new substrates identified roles in transcriptional be the of have identified II the SUMO to be in and cells. the sumoylation of and in in the is in we the sumoylation of and in II, our of an clustering of sumoylation in macromolecular complexes. of of in our clustering an of the regulation of by These a for sumoylation in processes by macromolecular proteins of be the sumoylation of is used a mechanism to the of in a sumoylation to an to a to the by the of the mechanism sumoylation cellular to our have to new SUMO substrates in Although in new targets the number of SUMO targets identified in the number of substrates identified in our of our sumoylation analysis the of to biological patterns and proteins are and a of new substrates to be and the of the sumoylation of factors the biological of for with with