AtAPY1 and AtAPY2 function as golgi-localized nucleoside diphosphatases in arabidopsis thaliana

Tsan Yu Chiu, Katy Christiansen, Ignacio Moreno, Jeemeng Lao, Dominique Loqué, Ariel Orellana, Joshua L. Heazlewood, Greg Clark, Stanley J. Roux

Resultado de la investigación: Article

19 Citas (Scopus)

Resumen

Nucleoside triphosphate diphosphohydrolases (NTPDases; apyrases) (EC 3.6.1.5) hydrolyze di-and triphosphate nucleotides, but not monophosphate nucleotides. They are categorized as E-type ATPases, have a broad divalent cation (Mg2+, Ca2+) requirement for activation and are insensitive to inhibitors of F-type, P-type and V-type ATPases. Among the seven NTPDases identified in Arabidopsis, only APYRASE 1 (AtAPY1) and APYRASE 2 (AtAPY2) have been previously characterized. In this work, either AtAPY1 or AtAPY2 tagged with C-terminal green fluorescent protein (GFP) driven by their respective native promoter can rescue the apy1 apy2 double knockout (apy1 apy2 dKO) successfully, and confocal microscopy reveals that these two Arabidopsis apyrases reside in the Golgi apparatus. In Saccharomyces cerevisiae, both AtAPY1 and AtAPY2 can complement the Golgilocalized GDA1 mutant, rescuing its aberrant protein glycosylation phenotype. In Arabidopsis, microsomes of the wild type show higher substrate preferences toward UDP compared with other NDP substrates. Loss-of-function Arabidopsis AtAPY1 mutants exhibit reduced microsomal UDPase activity, and this activity is even more significantly reduced in the loss-of-function AtAPY2 mutant and in the AtAPY1/AtAPY2 RNA interference (RNAi) technology repressor lines. Microsomes from wild-type plants also have detectable GDPase activity, which is significantly reduced in apy2 but not apy1 mutants. The GFP-tagged AtAPY1 or AtAPY2 constructs in the apy1 apy2 dKO plants can restore microsomal UDP/GDPase activity, confirming that they both also have functional competency. The cell walls of apy1, apy2 and the RNAi-silenced lines all have an increased composition of galactose, but the transport efficiency of UDP-galactose across microsomal membranes was not altered. Taken together, these results reveal that AtAPY1 and AtAPY2 are Golgi-localized nucleotide diphosphatases and are likely to have roles in regulating UDP/GDP concentrations in the Golgi lumen.

Idioma originalEnglish
Páginas (desde-hasta)1913-1925
Número de páginas13
PublicaciónPlant and Cell Physiology
Volumen53
N.º11
DOI
EstadoPublished - 1 nov 2012

Huella dactilar

nucleosides
apyrase
Nucleosides
Arabidopsis
Uridine Diphosphate
Arabidopsis thaliana
Apyrase
mutants
Nucleotides
nucleotides
microsomes
Microsomes
RNA Interference
Green Fluorescent Proteins
RNA interference
green fluorescent protein
galactose
Complement C2
Uridine Diphosphate Galactose
Vacuolar Proton-Translocating ATPases

ASJC Scopus subject areas

  • Plant Science
  • Physiology
  • Cell Biology

Citar esto

Chiu, Tsan Yu ; Christiansen, Katy ; Moreno, Ignacio ; Lao, Jeemeng ; Loqué, Dominique ; Orellana, Ariel ; Heazlewood, Joshua L. ; Clark, Greg ; Roux, Stanley J. / AtAPY1 and AtAPY2 function as golgi-localized nucleoside diphosphatases in arabidopsis thaliana. En: Plant and Cell Physiology. 2012 ; Vol. 53, N.º 11. pp. 1913-1925.
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title = "AtAPY1 and AtAPY2 function as golgi-localized nucleoside diphosphatases in arabidopsis thaliana",
abstract = "Nucleoside triphosphate diphosphohydrolases (NTPDases; apyrases) (EC 3.6.1.5) hydrolyze di-and triphosphate nucleotides, but not monophosphate nucleotides. They are categorized as E-type ATPases, have a broad divalent cation (Mg2+, Ca2+) requirement for activation and are insensitive to inhibitors of F-type, P-type and V-type ATPases. Among the seven NTPDases identified in Arabidopsis, only APYRASE 1 (AtAPY1) and APYRASE 2 (AtAPY2) have been previously characterized. In this work, either AtAPY1 or AtAPY2 tagged with C-terminal green fluorescent protein (GFP) driven by their respective native promoter can rescue the apy1 apy2 double knockout (apy1 apy2 dKO) successfully, and confocal microscopy reveals that these two Arabidopsis apyrases reside in the Golgi apparatus. In Saccharomyces cerevisiae, both AtAPY1 and AtAPY2 can complement the Golgilocalized GDA1 mutant, rescuing its aberrant protein glycosylation phenotype. In Arabidopsis, microsomes of the wild type show higher substrate preferences toward UDP compared with other NDP substrates. Loss-of-function Arabidopsis AtAPY1 mutants exhibit reduced microsomal UDPase activity, and this activity is even more significantly reduced in the loss-of-function AtAPY2 mutant and in the AtAPY1/AtAPY2 RNA interference (RNAi) technology repressor lines. Microsomes from wild-type plants also have detectable GDPase activity, which is significantly reduced in apy2 but not apy1 mutants. The GFP-tagged AtAPY1 or AtAPY2 constructs in the apy1 apy2 dKO plants can restore microsomal UDP/GDPase activity, confirming that they both also have functional competency. The cell walls of apy1, apy2 and the RNAi-silenced lines all have an increased composition of galactose, but the transport efficiency of UDP-galactose across microsomal membranes was not altered. Taken together, these results reveal that AtAPY1 and AtAPY2 are Golgi-localized nucleotide diphosphatases and are likely to have roles in regulating UDP/GDP concentrations in the Golgi lumen.",
keywords = "Arabidopsis, Functional complementation, Glycosylation, Golgi NTPDase",
author = "Chiu, {Tsan Yu} and Katy Christiansen and Ignacio Moreno and Jeemeng Lao and Dominique Loqu{\'e} and Ariel Orellana and Heazlewood, {Joshua L.} and Greg Clark and Roux, {Stanley J.}",
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Chiu, TY, Christiansen, K, Moreno, I, Lao, J, Loqué, D, Orellana, A, Heazlewood, JL, Clark, G & Roux, SJ 2012, 'AtAPY1 and AtAPY2 function as golgi-localized nucleoside diphosphatases in arabidopsis thaliana', Plant and Cell Physiology, vol. 53, n.º 11, pp. 1913-1925. https://doi.org/10.1093/pcp/pcs131

AtAPY1 and AtAPY2 function as golgi-localized nucleoside diphosphatases in arabidopsis thaliana. / Chiu, Tsan Yu; Christiansen, Katy; Moreno, Ignacio; Lao, Jeemeng; Loqué, Dominique; Orellana, Ariel; Heazlewood, Joshua L.; Clark, Greg; Roux, Stanley J.

En: Plant and Cell Physiology, Vol. 53, N.º 11, 01.11.2012, p. 1913-1925.

Resultado de la investigación: Article

TY - JOUR

T1 - AtAPY1 and AtAPY2 function as golgi-localized nucleoside diphosphatases in arabidopsis thaliana

AU - Chiu, Tsan Yu

AU - Christiansen, Katy

AU - Moreno, Ignacio

AU - Lao, Jeemeng

AU - Loqué, Dominique

AU - Orellana, Ariel

AU - Heazlewood, Joshua L.

AU - Clark, Greg

AU - Roux, Stanley J.

PY - 2012/11/1

Y1 - 2012/11/1

N2 - Nucleoside triphosphate diphosphohydrolases (NTPDases; apyrases) (EC 3.6.1.5) hydrolyze di-and triphosphate nucleotides, but not monophosphate nucleotides. They are categorized as E-type ATPases, have a broad divalent cation (Mg2+, Ca2+) requirement for activation and are insensitive to inhibitors of F-type, P-type and V-type ATPases. Among the seven NTPDases identified in Arabidopsis, only APYRASE 1 (AtAPY1) and APYRASE 2 (AtAPY2) have been previously characterized. In this work, either AtAPY1 or AtAPY2 tagged with C-terminal green fluorescent protein (GFP) driven by their respective native promoter can rescue the apy1 apy2 double knockout (apy1 apy2 dKO) successfully, and confocal microscopy reveals that these two Arabidopsis apyrases reside in the Golgi apparatus. In Saccharomyces cerevisiae, both AtAPY1 and AtAPY2 can complement the Golgilocalized GDA1 mutant, rescuing its aberrant protein glycosylation phenotype. In Arabidopsis, microsomes of the wild type show higher substrate preferences toward UDP compared with other NDP substrates. Loss-of-function Arabidopsis AtAPY1 mutants exhibit reduced microsomal UDPase activity, and this activity is even more significantly reduced in the loss-of-function AtAPY2 mutant and in the AtAPY1/AtAPY2 RNA interference (RNAi) technology repressor lines. Microsomes from wild-type plants also have detectable GDPase activity, which is significantly reduced in apy2 but not apy1 mutants. The GFP-tagged AtAPY1 or AtAPY2 constructs in the apy1 apy2 dKO plants can restore microsomal UDP/GDPase activity, confirming that they both also have functional competency. The cell walls of apy1, apy2 and the RNAi-silenced lines all have an increased composition of galactose, but the transport efficiency of UDP-galactose across microsomal membranes was not altered. Taken together, these results reveal that AtAPY1 and AtAPY2 are Golgi-localized nucleotide diphosphatases and are likely to have roles in regulating UDP/GDP concentrations in the Golgi lumen.

AB - Nucleoside triphosphate diphosphohydrolases (NTPDases; apyrases) (EC 3.6.1.5) hydrolyze di-and triphosphate nucleotides, but not monophosphate nucleotides. They are categorized as E-type ATPases, have a broad divalent cation (Mg2+, Ca2+) requirement for activation and are insensitive to inhibitors of F-type, P-type and V-type ATPases. Among the seven NTPDases identified in Arabidopsis, only APYRASE 1 (AtAPY1) and APYRASE 2 (AtAPY2) have been previously characterized. In this work, either AtAPY1 or AtAPY2 tagged with C-terminal green fluorescent protein (GFP) driven by their respective native promoter can rescue the apy1 apy2 double knockout (apy1 apy2 dKO) successfully, and confocal microscopy reveals that these two Arabidopsis apyrases reside in the Golgi apparatus. In Saccharomyces cerevisiae, both AtAPY1 and AtAPY2 can complement the Golgilocalized GDA1 mutant, rescuing its aberrant protein glycosylation phenotype. In Arabidopsis, microsomes of the wild type show higher substrate preferences toward UDP compared with other NDP substrates. Loss-of-function Arabidopsis AtAPY1 mutants exhibit reduced microsomal UDPase activity, and this activity is even more significantly reduced in the loss-of-function AtAPY2 mutant and in the AtAPY1/AtAPY2 RNA interference (RNAi) technology repressor lines. Microsomes from wild-type plants also have detectable GDPase activity, which is significantly reduced in apy2 but not apy1 mutants. The GFP-tagged AtAPY1 or AtAPY2 constructs in the apy1 apy2 dKO plants can restore microsomal UDP/GDPase activity, confirming that they both also have functional competency. The cell walls of apy1, apy2 and the RNAi-silenced lines all have an increased composition of galactose, but the transport efficiency of UDP-galactose across microsomal membranes was not altered. Taken together, these results reveal that AtAPY1 and AtAPY2 are Golgi-localized nucleotide diphosphatases and are likely to have roles in regulating UDP/GDP concentrations in the Golgi lumen.

KW - Arabidopsis

KW - Functional complementation

KW - Glycosylation

KW - Golgi NTPDase

UR - http://www.scopus.com/inward/record.url?scp=84869175511&partnerID=8YFLogxK

U2 - 10.1093/pcp/pcs131

DO - 10.1093/pcp/pcs131

M3 - Article

C2 - 23034877

AN - SCOPUS:84869175511

VL - 53

SP - 1913

EP - 1925

JO - Plant and Cell Physiology

JF - Plant and Cell Physiology

SN - 0032-0781

IS - 11

ER -