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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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Interactions of SUT2 and SUC2 as baits with other sucrose transporters detected by the split-ubiquitin system using LacZ as a reporter gene. Interaction of SUT2-CubPLV and SUC2-CubPLV as a bait was tested against various NubG-fusions of membrane proteins and soluble proteins as prey. Positive interaction was visualized by β-galactosidase expression in filter assays.
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Figure 3: Interactions of SUT2 and SUC2 as baits with other sucrose transporters detected by the split-ubiquitin system using LacZ as a reporter gene. Interaction of SUT2-CubPLV and SUC2-CubPLV as a bait was tested against various NubG-fusions of membrane proteins and soluble proteins as prey. Positive interaction was visualized by β-galactosidase expression in filter assays.

Mentions: The bait constructs were stably integrated into the genome of the reporter yeast strain. The transformed bait strains were subsequently transformed with the prey constructs. Both AtSUT2 as well as AtSUC2 were able to interact with themselves and with NubG-fusions of sucrose transporters AtSUT2, AtSUC2, and AtSUT4, but not with soluble NubG alone (Fig. 3). The results indicate that the three proteins can form oligomers. Interactions between sucrose transporters were confirmed using growth of the auxotrophic strain in absence of histidine. On control plates with histidine, yeast cells bearing the bait constructs AtSUC2-CubPLV or AtSUT2-CubPLV in combination with sucrose transporter prey constructs grow equally well as cells bearing bait constructs in combination with NubG only (Fig. 4A). In contrast, on plates without histidine, growth of yeast cells bearing interacting sucrose transporters is significantly stronger compared to cells bearing bait proteins in combination with NubG only (Fig. 4B). Thus, both markers detecting cleavage of the artificial transcription factor from the CubPLV upon protein-protein interaction can be used to study interactions between two membrane proteins.


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)

Interactions of SUT2 and SUC2 as baits with other sucrose transporters detected by the split-ubiquitin system using LacZ as a reporter gene. Interaction of SUT2-CubPLV and SUC2-CubPLV as a bait was tested against various NubG-fusions of membrane proteins and soluble proteins as prey. Positive interaction was visualized by β-galactosidase expression in filter assays.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Interactions of SUT2 and SUC2 as baits with other sucrose transporters detected by the split-ubiquitin system using LacZ as a reporter gene. Interaction of SUT2-CubPLV and SUC2-CubPLV as a bait was tested against various NubG-fusions of membrane proteins and soluble proteins as prey. Positive interaction was visualized by β-galactosidase expression in filter assays.
Mentions: The bait constructs were stably integrated into the genome of the reporter yeast strain. The transformed bait strains were subsequently transformed with the prey constructs. Both AtSUT2 as well as AtSUC2 were able to interact with themselves and with NubG-fusions of sucrose transporters AtSUT2, AtSUC2, and AtSUT4, but not with soluble NubG alone (Fig. 3). The results indicate that the three proteins can form oligomers. Interactions between sucrose transporters were confirmed using growth of the auxotrophic strain in absence of histidine. On control plates with histidine, yeast cells bearing the bait constructs AtSUC2-CubPLV or AtSUT2-CubPLV in combination with sucrose transporter prey constructs grow equally well as cells bearing bait constructs in combination with NubG only (Fig. 4A). In contrast, on plates without histidine, growth of yeast cells bearing interacting sucrose transporters is significantly stronger compared to cells bearing bait proteins in combination with NubG only (Fig. 4B). Thus, both markers detecting cleavage of the artificial transcription factor from the CubPLV upon protein-protein interaction can be used to study interactions between two membrane proteins.

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