Elucidation of the interactome of the sucrose transporter StSUT4: sucrose transport is connected to ethylene and calcium signalling

Abstract

Sucrose transporters of the SUT4 clade show dual targeting to both the plasma membrane as well as to the vacuole. Previous investigations revealed a role for the potato sucrose transporter StSUT4 in flowering, tuberization, shade avoidance response, and ethylene production. Down-regulation of StSUT4 expression leads to early flowering, tuberization under long days, far-red light insensitivity, and reduced diurnal ethylene production. Sucrose export from leaves was increased and a phase-shift of soluble sugar accumulation in source leaves was observed, arguing for StSUT4 to be involved in the entrainment of the circadian clock. Here, we show that StSUT4, whose transcripts are highly unstable and tightly controlled at the post-transcriptional level, connects components of the ethylene and calcium signalling pathway. Elucidation of the StSUT4 interactome using the split ubiquitin system helped to prove direct physical interaction between the sucrose transporter and the ethylene receptor ETR2, as well as with the calcium binding potato calmodulin-1 (PCM1) protein, and a calcium-load activated calcium channel. The impact of calcium ions on transport activity and dual targeting of the transporter was investigated in detail. For this purpose, a reliable esculin-based transport assay was established for SUT4-like transporters. Site-directed mutagenesis helped to identify a diacidic motif within the seventh transmembrane spanning domain that is essential for sucrose transport activity and targeting, but not required for calcium-dependent inhibition. A link between sucrose, calcium and ethylene signalling has been previously postulated with respect to pollen tube growth, shade avoidance response, or entrainment of the circadian clock. Here, we provide experimental evidence for the direct interconnection of these signalling pathways at the molecular level by direct physical interaction of the main players.

Keywords: Calcium binding; calcium channel; calcium inhibition; dual targeting; ethylene perception; protein-protein interaction; sub-cellular dynamics; sucrose transport.

Graphical Abstract

Fig. 1.

Sub-cellular localization of StSUT4. (A) Co-expression of a fluorescent StSUT4-YFP fusion protein (green) with a plasma membrane marker protein, CBL-OFP (orange) reveals no co-localization at the plasma membrane as previously observed under standard conditions (Garg et al. 2020). (B) StSUT4-RFP (red) is partially co-localized with a vacuolar marker protein, PTR2-YFP (green). Photographs were taken 3 d after infiltration with the Airyscan detector. Scale bar represents 10 µm.

Fig. 2.

StSUT4 protein interactions. (A) Confirmation of interaction of full length cDNAs of StETR2 and StPCM1 with the sucrose transporter StSUT4 by the split ubiquitin system; and (B-E) BiFC analysis. (A) Homodimer formation between StSUT4 in Nub and StSUT4 in Cub was taken as positive control. Full length cDNAs of ETR2 and PCP1 enable yeast strain L40ccU to grow on selective medium. Quantification of interaction strength was performed using ß-glucuronidase assay (blue colour). (B) Confirmation of StSUT4-ETR2 interaction in BiFC experiments. Green colour indicates YFP reconstitution and successful interaction. Autofluorescence of chlorophyll is shown in red. A single scan is shown. (C) Same cell as shown in (B) in a maximum projection of a z-stack, showing interaction in intracellular compartments. (D, E) Confirmation of StSUT4 interacting with StPCM1 was performed by BiFC in both orientations. Interaction takes place close to the plasma membrane. Scale bars represent 20 μm.

Fig. 3.

Esculin uptake assay. Establishment of a functional assay for StSUT4 in yeast cells using esculin as fluorescent sucrose analogue and determination of the optimal substrate concentration. (A) StSUT1, but not StSUT4 shows significantly higher esculin uptake at 1 mM substrate concentration than the empty vector control (pDR196). (B) StSUT4 and StSUT1 show significantly higher esculin uptake than the empty vector control (pDR196) at 8 mM substrate concentration after 1 h of incubation. Data are means ±SD; n=3. Student’s t-test was performed (*** P<0.001, **P<0.01 and *P<0.05).

Fig. 4.

pH dependence of esculin uptake by StSUT1 and StSUT4 in yeast cells. (A) StSUT1 shows a pH optimum in the highly acidic range (pH 3). (B) StSUT4 not only shows a reduced affinity towards its substrate but also a different pH dependence than StSUT1 with a pH optimum at pH 5. Data are means ±SD; n=4 independent measurements. (C) StSUT4-mediated esculin uptake in yeast cells in the presence of unlabelled sugars or no sugar. Competition studies revealed specificity of esculin uptake mediated by StSUT4 that is efficiently down-regulated in the presence of 20 mM sucrose. Data are shown as means ±SD, n=9 with three biological and three technical replicates, Student‘s t-test was performed (***P<0.001).

Fig. 5.

(A) StSUT4-mediated esculin uptake in yeast cells in the presence or absence of interaction partners. Co-expression of StSUT4-interaction partners StETR2 and StPCM1 reduces StSUT4-mediated esculin uptake. StSUT4 in pDR196 was co-expressed with the empty vector 112A1NE (red), StETR2 in 112A1NE (green) or StPCM1 in 112A1NE (blue). Data are means ±SD. Uptake was measured at pH 5. (B) Both sucrose transporters, StSUT1 as well as StSUT4 are efficiently inhibited by addition of 50 mM CaCl₂ after 1 h of incubation at pH 3. Student‘s t-test was performed (***P<0.001).

Fig. 6.

Quantification of StSUT4 transcripts and protein accumulation in response to various inhibitors or effectors. (A) Quantification of StSUT4 transcript amount via qPCR analysis using ubiquitin as a reference gene after treatment of source leaf material with either 50 mM CaCl₂, 50 mM MgCl₂ or water. Plant material was harvested at different time points (0–5 h).(B) qPCR analysis of StSUT4 transcript amount in source leaf material treated with the translational inhibitor cycloheximide (10 µM) for the indicated period of time (0–5 h) show a transient increase in transcript accumulation. Student’s t-test was performed to obtain significance values (n=4; ***P<0.001, **P<0.01, and *P<0.05). Ab: antibody. (C) Western blot analysis using the microsomal fraction of potato leaves incubated for several hours in water (water control), 10 µM of cycloheximide, or 50 mM CaCl₂. Incubation for several hours in 50 mM CaCl₂ show increased protein amount of StSUT4, but unchanged levels of StSUT1 protein. Cycloheximide effects on StSUT1 were published earlier (Kühn et al., 1997).

Fig. 7.

Changes in sub-cellular localization of StSUT1 and StSUT4 in response to CaCl₂ treatment. (A) Overnight treatment with 50 mM CaCl₂ at 21 °C induces increased vesicle formation of StSTUT4-YFP (shown in green) in the intracellular lumen of Nicotiana benthamiana epidermis cells, whereas water or EDTA do not. These effects are visible to a smaller extent if leaves were incubated in the cold (4 °C, upper row). YFP constructs (shown in green) were co-infiltrated with a PM marker (shown in orange) as before (Garg et al. 2020). (B) No internalization of StSUT1-YFP (green) from the plasma membrane was observed in response to overnight incubation of infiltrated leaves in 50 mM CaCl₂ at 21 °C. StSUT1 remains co-localized with the PM marker protein (orange) even in the presence of high amounts of calcium. Images were taken 3-4 d after infiltration using the Airyscan detector after the indicated time of incubation. Scale bar represents 10 μm.

Fig. 8.

(A) Split ubiquitin system with mutagenized StSUT4 constructs where the highly conserved DTD motif within the seventh transmembrane spanning domain is replaced by either GTG, NTN, or ETE, show the importance of this motif for efficient homodimerization. (B) StSUT4-mediated esculin uptake after mutagenesis of the conserved DTD motif at optimal pH (pH 5) and substrate concentration (8 mM). Mutagenesis of the DTD motif strongly affects StSUT4 transport activity. (C) Neither StSUT4, nor the DTD mutant constructs of StSUT4 are functional in the presence of 50 mM CaCl₂. Incubation time was 60 min. Note the difference in scale. Student’s t-test was performed to obtain significance values (n=4; ***P<0.001, **P<0.01, and *P<0.05).

Fig. 9.

Sub-cellular localization of StSU4 mutants. Co-localization experiments with StSUT4 mutant constructs co-infiltrated with either vacuolar (PTR2-YFP) or plasma membrane (CBL1-OFP) marker proteins. (A, B, D) None of the DTD mutant constructs could be co-localized with the vacuolar marker protein PTR2-YFP (arrow). (C, E) The StSUT4 DTD ETE-YFP and the StSUT4 DTD NTN-YFP construct show perfect overlap with the plasma membrane marker CBL1-OFP. Images were taken 3 d after infiltration using the Airyscan detector. Scale bars represent 10 µm (A, B, D, E) or 20 µm (C).