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Subcellular localization and topology of β(1→4)galactosyltransferase that elongates β(1→4)galactan side chains in rhamnogalacturonan I in potato

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Abstract

The subcellular localization and topology of rhamnogalacturonan I (RG-I) β(1→4)galactosyltransferase(s) (β[1→4]GalTs) from potato (Solanum tuberosum L.) were investigated. Using two-step discontinuous sucrose step gradients, galactosyltransferase (GalT) activity that synthesized 70%-methanol-insoluble products from UDP-[14C]Gal was detected in both the 0.5 M sucrose fraction and the 0.25/1.1 M sucrose interface. The former fraction contained mainly soluble proteins and the latter was enriched in Golgi vesicles that contained most of the UDPase activity, a Golgi marker. By gel-filtration analysis, products of 180–2,000 Da were found in the soluble fraction, whereas in the Golgi-enriched fraction the products were larger than 80 kDa and could be digested with rhamnogalacturonan lyase and β(1,4)endogalactanase to yield smaller rhamnogalacturonan oligomers, galactobiose and galactose. The endogalactanase requires β(1→4)galactans with at least three galactosyl residues for cleavage, indicating that the enzyme(s) present in the 0.25/1.1 M Suc interface transferred one or more galactosyl residues to pre-existing β(1→4)galactans producing RG-I side chains in total longer than a trimer. Thus, the β(1→4)GalT activity that elongates β(1→4)-linked galactan on RG-I was located in the Golgi apparatus. This β(1→4)GalT activity was not reduced after treatment of the Golgi vesicles with proteinase, but approximately 75% of the activity was lost after treatment with proteinase in the presence of Triton X-100. In addition, the β(1→4)GalT activity was recovered in the detergent phase after treatment of Golgi vesicles with Triton X-114. Taken together, these observations supported the view that the RG-I β(1→4)GalT that elongates β(1→4)galactan was mainly located in the Golgi apparatus and integrated into the membrane with its catalytic site facing the lumen.

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Abbreviations

GalT :

Galactosyltransferase

β(1→4)GalT :

β(1→4)-Galactosyltransferase

H + -ATPase :

Proton ATPase

HG :

Homogalacturonan

HSP70 :

ER resident Bip

mMDH :

Mitochondrial malate dehydrogenase

RG-I :

Rhamnogalacturonan I

RG-II :

Rhamnogalacturonan II

RGP :

Reversibly glycosylated polypeptide

RG-Lyase :

Rhamnogalacturonan lyase

Suc :

Sucrose

UDPase :

Uridine-5′-diphosphatase

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Acknowledgements

The authors thank Drs. Kanwarpal S. Dhugga (Pioneer Hi-Bred Intl., Iowa, USA), David J. Simpson (Carlsberg Laboratory, Denmark), Christine Gietl (Technical University München, Germany) and Michael Palmgren (Royal Veterinary and Agricultural University, Denmark) for the gift of antibodies against RGP, HSP70, mMDH and H+-ATPase, respectively. We also thank Dr. Kirk Schnorr (Novozymes A/S, Denmark) for the gift of recombinant RG-Lyase. Markus Pauly (Max-Planck Institute, Germany) and Henrik V. Scheller (Royal Veterinary and Agricultural University, Denmark) are thanked for helpful discussions. Helle M. Petersen (Pharmexa A/S, Denmark) and Winnie Dam are thanked for their skilful technical assistance. This work was supported by the Danish National Research Foundation.

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Authors and Affiliations

  1. Biotechnology Group, Danish Institute of Agricultural Sciences, Thorvaldsensvej 40. 8. 2., 1871, Frederiksberg C, Copenhagen, Denmark

    Naomi Geshi, Bodil Jørgensen & Peter Ulvskov

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  1. Naomi Geshi
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  2. Bodil Jørgensen
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  3. Peter Ulvskov
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Correspondence to Naomi Geshi.

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Geshi, N., Jørgensen, B. & Ulvskov, P. Subcellular localization and topology of β(1→4)galactosyltransferase that elongates β(1→4)galactan side chains in rhamnogalacturonan I in potato. Planta 218, 862–868 (2004). https://doi.org/10.1007/s00425-003-1168-3

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