Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase
The yeast Saccharomyces cerevisiae contains two known phosphoinositide 4-kinases (PI 4-kinases), which are encoded by PIK1 and STT4; both are essential. Pik1p is important for exocytic transport from the Golgi, whereas Stt4p plays a role in cell-wall integrity and cytoskeletal rearrangements. In the...
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Veröffentlicht in: | Biochemical journal 2003-04, Vol.371 (Pt 2), p.533-540 |
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description | The yeast Saccharomyces cerevisiae contains two known phosphoinositide 4-kinases (PI 4-kinases), which are encoded by PIK1 and STT4; both are essential. Pik1p is important for exocytic transport from the Golgi, whereas Stt4p plays a role in cell-wall integrity and cytoskeletal rearrangements. In the present study, we report that cells have a third PI 4-kinase activity encoded by LSB6, a protein identified previously in a two-hybrid screen as interacting with LAS17p. Although Pik1p and Stt4p are closely related members of the Type III class of PI 4-kinases, Lsb6p belongs to the distinct Type II class, based on its amino acid sequence, its sensitivity to inhibition by adenosine and its insensitivity to wortmannin. Lsb6p is the first fungal Type II enzyme cloned. The protein was expressed and purified from Sf9 cells and used to define kinetic parameters. As commonly observed for surface-active enzymes, activities varied both with substrate concentration and lipid/detergent molar ratios. Maximal activities of approx. 100 min(-1) were obtained at the PI/Triton X-100 ratio of 1:5. The K (m) value for ATP was 266 microM, intermediate between the values reported for mammalian Type II and III kinases. Epitope-tagged protein, expressed in yeast, was entirely particulate, and about half of it could be extracted with non-ionic detergent. Lsb6p-green fluorescent protein was found both on vacuolar membranes and on the plasma membrane, suggesting a role in endocytic or exocytic pathways. |
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Pik1p is important for exocytic transport from the Golgi, whereas Stt4p plays a role in cell-wall integrity and cytoskeletal rearrangements. In the present study, we report that cells have a third PI 4-kinase activity encoded by LSB6, a protein identified previously in a two-hybrid screen as interacting with LAS17p. Although Pik1p and Stt4p are closely related members of the Type III class of PI 4-kinases, Lsb6p belongs to the distinct Type II class, based on its amino acid sequence, its sensitivity to inhibition by adenosine and its insensitivity to wortmannin. Lsb6p is the first fungal Type II enzyme cloned. The protein was expressed and purified from Sf9 cells and used to define kinetic parameters. As commonly observed for surface-active enzymes, activities varied both with substrate concentration and lipid/detergent molar ratios. Maximal activities of approx. 100 min(-1) were obtained at the PI/Triton X-100 ratio of 1:5. The K (m) value for ATP was 266 microM, intermediate between the values reported for mammalian Type II and III kinases. Epitope-tagged protein, expressed in yeast, was entirely particulate, and about half of it could be extracted with non-ionic detergent. Lsb6p-green fluorescent protein was found both on vacuolar membranes and on the plasma membrane, suggesting a role in endocytic or exocytic pathways.</description><identifier>ISSN: 0264-6021</identifier><identifier>EISSN: 1470-8728</identifier><identifier>DOI: 10.1042/bj20021407</identifier><identifier>PMID: 12523934</identifier><language>eng</language><publisher>England</publisher><subject>1-Phosphatidylinositol 4-Kinase - genetics ; 1-Phosphatidylinositol 4-Kinase - isolation & purification ; 1-Phosphatidylinositol 4-Kinase - metabolism ; adenosine ; Base Sequence ; Cloning, Molecular ; Kinetics ; Lsb6 protein ; Molecular Sequence Data ; phosphoinositide 4-kinase ; Pik1 protein ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae - enzymology ; Saccharomyces cerevisiae Proteins - genetics ; Saccharomyces cerevisiae Proteins - isolation & purification ; Saccharomyces cerevisiae Proteins - metabolism ; Stt4 protein ; Substrate Specificity</subject><ispartof>Biochemical journal, 2003-04, Vol.371 (Pt 2), p.533-540</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c471t-e5246737457656e828687bdb2eb85bab913fc1e0d8486117cc7486576817e86b3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223293/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223293/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12523934$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Shelton, Shary N</creatorcontrib><creatorcontrib>Barylko, Barbara</creatorcontrib><creatorcontrib>Binns, Derk D</creatorcontrib><creatorcontrib>Horazdovsky, Bruce F</creatorcontrib><creatorcontrib>Albanesi, Joseph P</creatorcontrib><creatorcontrib>Goodman, Joel M</creatorcontrib><title>Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase</title><title>Biochemical journal</title><addtitle>Biochem J</addtitle><description>The yeast Saccharomyces cerevisiae contains two known phosphoinositide 4-kinases (PI 4-kinases), which are encoded by PIK1 and STT4; both are essential. Pik1p is important for exocytic transport from the Golgi, whereas Stt4p plays a role in cell-wall integrity and cytoskeletal rearrangements. In the present study, we report that cells have a third PI 4-kinase activity encoded by LSB6, a protein identified previously in a two-hybrid screen as interacting with LAS17p. Although Pik1p and Stt4p are closely related members of the Type III class of PI 4-kinases, Lsb6p belongs to the distinct Type II class, based on its amino acid sequence, its sensitivity to inhibition by adenosine and its insensitivity to wortmannin. Lsb6p is the first fungal Type II enzyme cloned. The protein was expressed and purified from Sf9 cells and used to define kinetic parameters. As commonly observed for surface-active enzymes, activities varied both with substrate concentration and lipid/detergent molar ratios. Maximal activities of approx. 100 min(-1) were obtained at the PI/Triton X-100 ratio of 1:5. The K (m) value for ATP was 266 microM, intermediate between the values reported for mammalian Type II and III kinases. Epitope-tagged protein, expressed in yeast, was entirely particulate, and about half of it could be extracted with non-ionic detergent. Lsb6p-green fluorescent protein was found both on vacuolar membranes and on the plasma membrane, suggesting a role in endocytic or exocytic pathways.</description><subject>1-Phosphatidylinositol 4-Kinase - genetics</subject><subject>1-Phosphatidylinositol 4-Kinase - isolation & purification</subject><subject>1-Phosphatidylinositol 4-Kinase - metabolism</subject><subject>adenosine</subject><subject>Base Sequence</subject><subject>Cloning, Molecular</subject><subject>Kinetics</subject><subject>Lsb6 protein</subject><subject>Molecular Sequence Data</subject><subject>phosphoinositide 4-kinase</subject><subject>Pik1 protein</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>Saccharomyces cerevisiae</subject><subject>Saccharomyces cerevisiae - enzymology</subject><subject>Saccharomyces cerevisiae Proteins - genetics</subject><subject>Saccharomyces cerevisiae Proteins - isolation & purification</subject><subject>Saccharomyces cerevisiae Proteins - metabolism</subject><subject>Stt4 protein</subject><subject>Substrate Specificity</subject><issn>0264-6021</issn><issn>1470-8728</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU9LAzEQxYMotlYvfgDZkwdhNf82yV4EKVULBQ_Wc8hmpzZ1d7Mm20K_vSstVk_CDDPwfjwePIQuCb4lmNO7YkUxpoRjeYSGhEucKknVMRpiKngqemmAzmJcYdwzHJ-iAaEZZTnjQzR5NdYuTfD11kJMLATYuOgMJNY3nXFNTEwy37aQTKdJu_SxX9f46DpXQsLTD9eYCOfoZGGqCBf7O0Jvj5P5-DmdvTxNxw-z1HJJuhQyyoVkkmdSZAIUVULJoiwoFCorTJETtrAEcKm4EoRIa2X_9LAiEpQo2Ajd73zbdVFDaaHpgql0G1xtwlZ74_RfpXFL_e43mlDKaM56g-u9QfCfa4idrl20UFWmAb-OWjLC84zKf0GS00ywfkboZgfa4GMMsPhJQ7D-rkcf6unhq9_5D-i-D_YFS2KKGQ</recordid><startdate>20030415</startdate><enddate>20030415</enddate><creator>Shelton, Shary N</creator><creator>Barylko, Barbara</creator><creator>Binns, Derk D</creator><creator>Horazdovsky, Bruce F</creator><creator>Albanesi, Joseph P</creator><creator>Goodman, Joel M</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>M7N</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20030415</creationdate><title>Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase</title><author>Shelton, Shary N ; Barylko, Barbara ; Binns, Derk D ; Horazdovsky, Bruce F ; Albanesi, Joseph P ; Goodman, Joel M</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c471t-e5246737457656e828687bdb2eb85bab913fc1e0d8486117cc7486576817e86b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2003</creationdate><topic>1-Phosphatidylinositol 4-Kinase - genetics</topic><topic>1-Phosphatidylinositol 4-Kinase - isolation & purification</topic><topic>1-Phosphatidylinositol 4-Kinase - metabolism</topic><topic>adenosine</topic><topic>Base Sequence</topic><topic>Cloning, Molecular</topic><topic>Kinetics</topic><topic>Lsb6 protein</topic><topic>Molecular Sequence Data</topic><topic>phosphoinositide 4-kinase</topic><topic>Pik1 protein</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - metabolism</topic><topic>Saccharomyces cerevisiae</topic><topic>Saccharomyces cerevisiae - enzymology</topic><topic>Saccharomyces cerevisiae Proteins - genetics</topic><topic>Saccharomyces cerevisiae Proteins - isolation & purification</topic><topic>Saccharomyces cerevisiae Proteins - metabolism</topic><topic>Stt4 protein</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Shelton, Shary N</creatorcontrib><creatorcontrib>Barylko, Barbara</creatorcontrib><creatorcontrib>Binns, Derk D</creatorcontrib><creatorcontrib>Horazdovsky, Bruce F</creatorcontrib><creatorcontrib>Albanesi, Joseph P</creatorcontrib><creatorcontrib>Goodman, Joel M</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Biochemical journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Shelton, Shary N</au><au>Barylko, Barbara</au><au>Binns, Derk D</au><au>Horazdovsky, Bruce F</au><au>Albanesi, Joseph P</au><au>Goodman, Joel M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase</atitle><jtitle>Biochemical journal</jtitle><addtitle>Biochem J</addtitle><date>2003-04-15</date><risdate>2003</risdate><volume>371</volume><issue>Pt 2</issue><spage>533</spage><epage>540</epage><pages>533-540</pages><issn>0264-6021</issn><eissn>1470-8728</eissn><abstract>The yeast Saccharomyces cerevisiae contains two known phosphoinositide 4-kinases (PI 4-kinases), which are encoded by PIK1 and STT4; both are essential. Pik1p is important for exocytic transport from the Golgi, whereas Stt4p plays a role in cell-wall integrity and cytoskeletal rearrangements. In the present study, we report that cells have a third PI 4-kinase activity encoded by LSB6, a protein identified previously in a two-hybrid screen as interacting with LAS17p. Although Pik1p and Stt4p are closely related members of the Type III class of PI 4-kinases, Lsb6p belongs to the distinct Type II class, based on its amino acid sequence, its sensitivity to inhibition by adenosine and its insensitivity to wortmannin. Lsb6p is the first fungal Type II enzyme cloned. The protein was expressed and purified from Sf9 cells and used to define kinetic parameters. As commonly observed for surface-active enzymes, activities varied both with substrate concentration and lipid/detergent molar ratios. Maximal activities of approx. 100 min(-1) were obtained at the PI/Triton X-100 ratio of 1:5. The K (m) value for ATP was 266 microM, intermediate between the values reported for mammalian Type II and III kinases. Epitope-tagged protein, expressed in yeast, was entirely particulate, and about half of it could be extracted with non-ionic detergent. Lsb6p-green fluorescent protein was found both on vacuolar membranes and on the plasma membrane, suggesting a role in endocytic or exocytic pathways.</abstract><cop>England</cop><pmid>12523934</pmid><doi>10.1042/bj20021407</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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subjects | 1-Phosphatidylinositol 4-Kinase - genetics 1-Phosphatidylinositol 4-Kinase - isolation & purification 1-Phosphatidylinositol 4-Kinase - metabolism adenosine Base Sequence Cloning, Molecular Kinetics Lsb6 protein Molecular Sequence Data phosphoinositide 4-kinase Pik1 protein Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Saccharomyces cerevisiae Saccharomyces cerevisiae - enzymology Saccharomyces cerevisiae Proteins - genetics Saccharomyces cerevisiae Proteins - isolation & purification Saccharomyces cerevisiae Proteins - metabolism Stt4 protein Substrate Specificity |
title | Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase |
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