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Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system.

Schulze WX, Reinders A, Ward J, Lalonde S, Frommer WB - BMC Biochem. (2003)

Bottom Line: The interactions are specific, since a potassium channel and a glucose transporter did not show interaction with sucrose transporters.Also the biosynthetic and metabolizing enzymes, sucrose phosphate phosphatase and sucrose synthase, which were found to be at least in part bound to the plasma membrane, did not specifically interact with sucrose transporters.The biochemical approaches are required to confirm the in planta interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Physiology, Zentrum für Molekularbiologie der Pflanzen (ZMBP), Universität Tübingen, Auf der Morgenstelle1, D-72076 Tübingen, Germany. waltraud@bmb.sdu.dk

ABSTRACT

Background: The Arabidopsis genome contains nine sucrose transporter paralogs falling into three clades: SUT1-like, SUT2 and SUT4. The carriers differ in their kinetic properties. Many transport proteins are known to exist as oligomers. The yeast-based split ubiquitin system can be used to analyze the ability of membrane proteins to interact.

Results: Promoter-GUS fusions were used to analyze the cellular expression of the three transporter genes in transgenic Arabidopsis plants. All three fusion genes are co-expressed in companion cells. Protein-protein interactions between Arabidopsis sucrose transporters were tested using the split ubiquitin system. Three paralogous sucrose transporters are capable of interacting as either homo- or heteromers. The interactions are specific, since a potassium channel and a glucose transporter did not show interaction with sucrose transporters. Also the biosynthetic and metabolizing enzymes, sucrose phosphate phosphatase and sucrose synthase, which were found to be at least in part bound to the plasma membrane, did not specifically interact with sucrose transporters.

Conclusions: The split-ubiquitin system provides a powerful tool to detect potential interactions between plant membrane proteins by heterologous expression in yeast, and can be used to screen for interactions with membrane proteins as baits. Like other membrane proteins, the Arabidopsis sucrose transporters are able to form oligomers. The biochemical approaches are required to confirm the in planta interaction.

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Functionality of NubG-AtSUC2 and NubG-AtSUT4 fusion proteins. Growth of yeast strain SUSY7/ura3 bearing respective fusion proteins or the empty NubG-vector on medium containing 2% glucose or 2% sucrose as the sole carbon source after four days of growth at 28°C.
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Figure 5: Functionality of NubG-AtSUC2 and NubG-AtSUT4 fusion proteins. Growth of yeast strain SUSY7/ura3 bearing respective fusion proteins or the empty NubG-vector on medium containing 2% glucose or 2% sucrose as the sole carbon source after four days of growth at 28°C.

Mentions: To confirm the targeting of the NubG-fusion proteins to the plasma membrane and to show their functionality, NubG-AtSUC2 and NubG-AtSUT4 were tested for their ability to complement growth of the yeast strain SUSY7/ura3 on sucrose [11,30]. In both cases, the N-terminal fusion of NubG to the sucrose transporters did not impair their transport function, as demonstrated by the growth of SUSY7/ura3 containing the respective fusion proteins on sucrose as the sole carbon source (Fig. 5). Kinetic analysis (data not shown) revealed no significant difference between sucrose affinity of NubG-fusion proteins and published sucrose affinities [3,10].


Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system.

Schulze WX, Reinders A, Ward J, Lalonde S, Frommer WB - BMC Biochem. (2003)

Functionality of NubG-AtSUC2 and NubG-AtSUT4 fusion proteins. Growth of yeast strain SUSY7/ura3 bearing respective fusion proteins or the empty NubG-vector on medium containing 2% glucose or 2% sucrose as the sole carbon source after four days of growth at 28°C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC153512&req=5

Figure 5: Functionality of NubG-AtSUC2 and NubG-AtSUT4 fusion proteins. Growth of yeast strain SUSY7/ura3 bearing respective fusion proteins or the empty NubG-vector on medium containing 2% glucose or 2% sucrose as the sole carbon source after four days of growth at 28°C.
Mentions: To confirm the targeting of the NubG-fusion proteins to the plasma membrane and to show their functionality, NubG-AtSUC2 and NubG-AtSUT4 were tested for their ability to complement growth of the yeast strain SUSY7/ura3 on sucrose [11,30]. In both cases, the N-terminal fusion of NubG to the sucrose transporters did not impair their transport function, as demonstrated by the growth of SUSY7/ura3 containing the respective fusion proteins on sucrose as the sole carbon source (Fig. 5). Kinetic analysis (data not shown) revealed no significant difference between sucrose affinity of NubG-fusion proteins and published sucrose affinities [3,10].

Bottom Line: The interactions are specific, since a potassium channel and a glucose transporter did not show interaction with sucrose transporters.Also the biosynthetic and metabolizing enzymes, sucrose phosphate phosphatase and sucrose synthase, which were found to be at least in part bound to the plasma membrane, did not specifically interact with sucrose transporters.The biochemical approaches are required to confirm the in planta interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Physiology, Zentrum für Molekularbiologie der Pflanzen (ZMBP), Universität Tübingen, Auf der Morgenstelle1, D-72076 Tübingen, Germany. waltraud@bmb.sdu.dk

ABSTRACT

Background: The Arabidopsis genome contains nine sucrose transporter paralogs falling into three clades: SUT1-like, SUT2 and SUT4. The carriers differ in their kinetic properties. Many transport proteins are known to exist as oligomers. The yeast-based split ubiquitin system can be used to analyze the ability of membrane proteins to interact.

Results: Promoter-GUS fusions were used to analyze the cellular expression of the three transporter genes in transgenic Arabidopsis plants. All three fusion genes are co-expressed in companion cells. Protein-protein interactions between Arabidopsis sucrose transporters were tested using the split ubiquitin system. Three paralogous sucrose transporters are capable of interacting as either homo- or heteromers. The interactions are specific, since a potassium channel and a glucose transporter did not show interaction with sucrose transporters. Also the biosynthetic and metabolizing enzymes, sucrose phosphate phosphatase and sucrose synthase, which were found to be at least in part bound to the plasma membrane, did not specifically interact with sucrose transporters.

Conclusions: The split-ubiquitin system provides a powerful tool to detect potential interactions between plant membrane proteins by heterologous expression in yeast, and can be used to screen for interactions with membrane proteins as baits. Like other membrane proteins, the Arabidopsis sucrose transporters are able to form oligomers. The biochemical approaches are required to confirm the in planta interaction.

Show MeSH
Related in: MedlinePlus