Apple sucrose transporter SUT1 and sorbitol transporter SOT6 interact with cytochrome b5 to regulate their affinity for substrate sugars

Abstract

Sugar transporters are central machineries to mediate cross-membrane transport of sugars into the cells, and sugar availability may serve as a signal to regulate the sugar transporters. However, the mechanisms of sugar transport regulation by signal sugar availability remain unclear in plant and animal cells. Here, we report that a sucrose transporter, MdSUT1, and a sorbitol transporter, MdSOT6, both localized to plasma membrane, were identified from apple (Malus domestica) fruit. Using a combination of the split-ubiquitin yeast two-hybrid, immunocoprecipitation, and bimolecular fluorescence complementation assays, the two distinct sugar transporters were shown to interact physically with an apple endoplasmic reticulum-anchored cytochrome b5 MdCYB5 in vitro and in vivo. In the yeast systems, the two different interaction complexes function to up-regulate the affinity of the sugar transporters, allowing cells to adapt to sugar starvation. An Arabidopsis (Arabidopsis thaliana) homolog of MdCYB5, AtCYB5-A, also interacts with the two sugar transporters and functions similarly. The point mutations leucine-73 --> proline in MdSUT1 and leucine-117 --> proline in MdSOT6, disrupting the bimolecular interactions but without significantly affecting the transporter activities, abolish the stimulating effects of the sugar transporter-cytochrome b5 complex on the affinity of the sugar transporters. However, the yeast (Saccharomyces cerevisiae) cytochrome b5 ScCYB5, an additional interacting partner of the two plant sugar transporters, has no function in the regulation of the sugar transporters, indicating that the observed biological functions in the yeast systems are specific to plant cytochrome b5s. These findings suggest a novel mechanism by which the plant cells tailor sugar uptake to the surrounding sugar availability.

Figure 1.

Identification of MdSUT1 and MdSOT6 as functional sugar transporters. A, Expression of MdSUT1 in the yeast strain SUSY7/ura3 allows growth on Suc as the sole carbon source. SUSY7/ura3 yeast cells were transformed with the empty pDR196 vector (indicated by pDR196; top panels) or MdSUT1 in the vector pDR196 (indicated by MdSUT1; bottom panels). Yeast cells of 8 μL were spotted at three concentrations (OD₆₀₀ = 0.1, 0.05, and 0.01) on synthetic minimal media containing 2% (w/v) Suc or Glc and 20 mg L⁻¹ Trp at pH 5.0 and grown at 30°C for 4 d for observations. The assays were repeated three times with the same results. B, Time course of Suc uptake by SUSY7/ura3 expressing MdSUT1. SUSY7/ura3 yeast transformed with MdSUT1 in pDR196 and empty pDR196 vector were assayed for [¹⁴C]Suc uptake at 0.4 mm Suc and pH 5.0. C, Suc uptake kinetics of SUSY7/ura3 expressing MdSUT1. Transport assays were performed at pH 5.0, and uptake rates were plotted against Suc concentrations in the assays. D, Expression of MdSOT6 in the yeast strain RS453 allows growth on sorbitol as the sole carbon source. RS453 yeast cells were transformed with the empty pDR196 vector (indicated by pDR196; top panels) or MdSOT6 in the vector pDR196 (indicated by MdSOT6; bottom panels). Cells of 8 μL were spotted at three concentrations (OD₆₀₀ = 0.1, 0.05, and 0.01) on synthetic minimal media containing 2% (w/v) sorbitol or Glc and 20 mg L⁻¹ adenine, Leu, Trp, and His at pH 5.0 and grown at 30°C for 4 d for observations. The assays were repeated three times with the same results. E, Time course of sorbitol uptake by RS453 expressing MdSOT6. RS453 yeast transformed with MdSOT6 in pDR196 or empty pDR196 vector were assayed for [³H]sorbitol uptake at 0.25 mm sorbitol and pH 4.5. F, Sorbitol uptake kinetics of RS453 expressing MdSOT6. Transport assays were performed at pH 4.5, and uptake rates were plotted against sorbitol concentrations in the assays. Values in B, C, E, and F are means ± se (n = 3).

Figure 2.

A putative cytochrome b5 MdCYB5 interacts with the MdSUT1 and MdSOT6 in the yeast split-ubiquitin system. A, Schematic presentation of the constructs for the two-hybrid analysis in the split-ubiquitin system. (a) Construct MdSUT1- or MdSOT6-CubPLV in the pTMBV4 bait vector. The restriction sites XbaI-NcoI were used for MdSUT1 and XbaI-StuI were used for MdSOT6. (b) Construct NubG-MdCYB5 in the pDL2 prey vector. (c) Construct NubG-cDNA in the pDL2 prey vector, where cDNA represents apple fruit cDNA. ADH1, Alcohol dehydrogenase1 promoter; Cub, C terminus of ubiquitin; CYC1, cytochrome c terminator; NubG, the mutated N-terminal (with mutation Ile-13→Gly) subdomain of ubiquitin; PLV, an artificial transcription factor consisting mainly of Lex A and VP16; TEF1, transcription elongation factor1 promoter. Bar = 1,000 bp for length. The restriction sites are indicated above the scheme. See “Materials and Methods” for detailed information. B, Interactions of the different MdSUT1 truncations from the C terminus of MdSUT1 with MdCYB5 (prey construct NubG-CYB5). The numbers 1-n after SUT1 indicate the amino acid numbers from the N-terminal first amino acid to the truncated site (n) in the protein (left panel). The schematic structures of the MdSUT1 (middle-left panel) are presented: the black portions indicate the sequences retained and the gray portions indicate the sequences removed. The middle-right (−His growth) panel indicates the transformant yeast growth in the His-deficient medium, with symbol ++ indicating good status of growth and − indicating no apparent growth of the yeast cells. The right panel (β-Gal) indicates the β-gal activity developed on filter papers. The asterisk indicates the MdSUT1 with a point mutation at Leu-73→Pro. All other symbols or abbreviations are as described in A, and the single + indicates a decreased growth compared with the growth expressed by ++. C, Interactions of the different MdSOT6 truncations from the C terminus of MdSOT6 with MdCYB5 (prey construct NubG-CYB5). The numbers 1-n after SOT6 indicate the amino acid numbers from the N-terminal first amino acid to the truncated site (n) in the protein (left panel). The schematic structures of the MdSOT6 (middle-left panel) are presented: the black portions indicate the sequences retained and the gray portions indicate the sequences removed. The asterisk indicates the MdSOT6 with a point mutation. All other symbols or abbreviations are as described in A and B. D, Interactions of the different truncations of MdCYB5 with MdSOT6 (bait construct SOT6-CubPLV) with MdCYB5. The numbers after CYB5 indicate the amino acid numbers of the beginning and the end of the fragments left after truncation (left panel). The schematic structures of the MdCYB5 (middle-left panel) are presented: the black portions indicate the sequences retained and the gray portions indicate the sequences removed. All other symbols or abbreviations are as described in A to C. β-Gal activity was quantified (right panel; relative units).

Figure 3.

Interaction of MdCYB5 with the MdSUT1 and MdSOT6 identified by pull-down assays. A, MdCYB5 coimmunoprecipitates with MdSUT1 in the yeast split-ubiquitin system. The total proteins were prepared from the yeast cells transformed with different constructs, including the pTMBV4 vector harboring MdSUT1 (pTMBV4-SUT1), the pDL2 vector harboring MdCYB5 (pDL2-CYB5), the pDL2-Alg5 control vector, the pMBV4-Alg5 control vector plus pDL2-CYB5 (pMBV4-Alg5+pDL2-CYB5), and pMBV4-SUT1 plus pDL2-CYB5. The total proteins were immunoblotted with the antiserum against the tag VP16 for MdSUT1 (a) or with the antiserum against the tag HA for CYB5 (b) for two positive controls. The two immunodetected bands migrating as higher molecular mass proteins should be the complex MdSUT1-VP16, and that migrating at low molecular mass should be VP16. The total proteins were precipitated with the anti-VP16 serum (IP: antiVP16), and the immunoprecipitates were blotted with the anti-VP16 serum as the third positive control [Blot: antiVP16 (c)] or anti-HA serum [Blot: antiHA (d)]. The immunodetected bands with higher molecular mass indicate the complex MdSUT1-VP16, and that with lower molecular mass indicates VP16 (c). The immunoprecipitates obtained with the anti-HA serum from the total proteins were blotted with either the anti-HA serum [as the fourth positive control (e)] or the anti-VP16 serum (f). B, MdCYB5 coimmunoprecipitates with MdSUT1 in the membrane proteins from apple. The membrane proteins were isolated from the total proteins and immunoblotted with the anti-MdCYB5 serum (Blot: antiCYB5) or anti-MdSUT1^CL serum (Blot: antiSUT1^CL) as two positive controls. The membrane proteins were precipitated with anti-MdSUT1^CL serum (IP: antiMdSUT1^CL), and the immunoprecipitates were blotted with the anti-MdCYB5 serum, which detected a 15-kD MdCYB5 signal (left panel). The immunoprecipitates obtained with the anti-MdCYB5 serum from the membrane proteins were blotted with the anti-MdSUT1^CL serum, which detected a 56-kD MdSUT1 signal (right panel). In both cases, the immunoprecipitates obtained with the preimmune serum from the total proteins (IP: preimmune serum) served as negative controls. C, MdCYB5 coimmunoprecipitates with MdSOT6 in the yeast split-ubiquitin system. The total proteins were prepared from the yeast cells transformed with different constructs, including the pTMBV4 vector harboring MdSOT6 (pTMBV4-SOT6), the pDL2 vector harboring MdCYB5 (pDL2-CYB5), and pTMBV4-SOT6 plus pDL2-CYB5 (pTMBV4-SOT6+pDL2-CYB5). The total proteins and the immunoprecipitates isolated from the total proteins with either the anti-HA serum (IP: antiHA) or the anti-VP16 serum (IP: antiVP16) were immunoblotted with the anti-VP16 serum (Blot: antiVP16; top panel) or with the anti-HA serum (Blot: antiHA; bottom panel). D, MdCYB5 coimmunoprecipitates with MdSOT6 in the membrane proteins from apple. The membrane proteins and the immunoprecipitates obtained from the membrane proteins with either the anti-MdSOT6^CL serum (IP: anti-MdSOT6^CL) or the anti-MdCYB5 serum (IP: antiCYB5) were immunoblotted with the anti-MdSOT6^CL serum (Blot: antiSOT6^CL; top panel shows a 54-kD immunosignal detected) or anti-MdCYB5 serum (Blot: antiCYB5; the bottom panel shows a 15-kD immunosignal detected). The immunoprecipitates obtained with the preimmune serum from the membrane proteins (IP: preimmune serum) served as negative controls.

Figure 4.

In vivo interaction of MdCYB5 with MdSUT1 and MdSOT6 in the BiFC system. The laser-scanning confocal microscopy images show fluorescence (indicated by YFP) and merged images of the double transgenic cells with the YFP_N-MdCYB5 and MdSUT1-YFP_C fusions (YFP_N-CYB5/SUT1-YFP_C) or with the YFP_N-CYB5/SOT6-YFP_C construct pair. The wild-type MdSUT1 and MdSOT6 were replaced in the above-mentioned constructs by the mutated MdSUT1_P (Leu-73→Pro; YFP_N-CYB5/SUT1_P-YFP_C) and mutated MdSOT6_P (Leu-117→Pro; YFP_N-CYB5/SOT6_P-YFP_C), respectively, which were used to transform cells that served as negative controls. The construct pairs YFP_N-CYB5/YFP_C, YFP_N/SUT1-YFP_C, and YFP_N/SOT6-YFP_C were also used as negative controls to transform the protoplasts. The chlorophyll autofluorescence (Auto-) and the bright-field images are also presented.

Figure 5.

Localization of MdSUT1, MdSOT6, and MdCYB5 in cells. A, Transient expression of MdSUT1-GFP (and its mutated form MdSUT1_P), MdSOT6-GFP (and its mutated form MdSOT6_P), and MdCYB5-GFP fusion proteins in the Arabidopsis protoplasts. The laser-scanning confocal microscopy images show the MdSUT1 (indicated by SUT1-GFP), MdSOT6 (SOT6-GFP), GFP-MdCYB5 (GFP-CYB5), MdSUT1_P (SUT1_P-GFP), and MdSOT6_P (SOT6_P-GFP) fluorescence (GFP) and the merged images. The chlorophyll autofluorescence (Auto-) and the bright-field images are also presented. Note that the mutation of MdSUT1_P did not change the plasma membrane localization of MdSUT1, and the mutation of MdSOT6_P did not change the plasma membrane localization of MdSOT6. B to E, Immunogold labeling of MdCYB5 in apple fruit cells. The protein reacting with anti-MdCYB5^N (visualized by immunogold particles) mainly resides on the ER (B and D). A blowup (C) of the area from B shows more clearly the ER localization of MdCYB5; note that some of the ER hosting MdCYB5 immunoparticles distribute near the plasma membrane (PM), which are shown by arrows. D shows more clearly the distribution of MdCYB5-hosting ER around the plasma membrane (indicated by arrows). No substantial signal was detected in other cellular compartments (B–D) such as the cell wall (CW), mitochondrion (M), and cytoplasm. No substantial gold particles were found in the control cells without antiserum treatment (E). Bars = 0.5 μm.

Figure 6.

Alignment of MdCYB5 and its closed homologs, and homology of plant cytochrome b5s. A, Deduced amino acid sequence alignment of AtCYB5, OeCYB5, MdCYB5, and ScCYB5, showing the presence of conserved features and including the predicted heme-binding domain (underlined in blue), heme-binding signature (violet line above), C-terminal transmembrane domain (underlined in red), and C-terminal polar sequence (underlined in pink) among the three plant CYB5s (AtCYB5-A, OeCYB5, and MdCYB5). The C-terminal transmembrane domain of the yeast ScCYB5 is underlined in green. Numbers at right indicate numbers of amino acid residues in the predicted sequences. Gaps, indicated by dots, were induced to maximize alignment. Identical amino acid residues between two or more CYB5s are indicated by white letters on a black background, and similar residues are indicated by black letters on a gray background. B, Homology tree of plant cytochrome b5. The prefixes of the CYB5 names represent the following plant species: Ac, Anana comosus; At, Arabidopsis thaliana; Bo, Brassica oleracea; Cr, Cuscuta reflexa; Hs, Homo sapiens; Md, Malus domestica; Mt, Medicago truncatula; Nt, Nicotiana tabacum; Os, Oryza sativa; Pt, Pan troglodytes; Rn, Rattus norvegicus; Vf, Vernicia fordii; Vv, Vitis vinifera. Percentages of amino acid sequence identity are indicated. The corresponding accession numbers of these cytochrome b5s are given at the end of “Materials and Methods.”

Figure 7.

Both the Arabidopsis CYB5-A (AtCYB5-A) and yeast CYB5 (ScCYB5) interact with MdSUT1 and MdSOT6. A, Interactions of AtCYB5-A and ScCYB5 with MdSUT1 and MdSOT6 tested by β-gal activity through filter assays in the yeast split-ubiquitin system. The MdSUT1 and MdSOT6 were recombined into the bait vector pTMBV4, and the CYB5s, including MdCYB5, AtCYB5-A, and ScCYB5, were recombined into the prey vector pDL-Nx or pDL-xN (where x indicates the CYB5 cDNAs and N denotes the mutated N terminus of the subdomain of ubiquitin). The yeast cells were cotransformed with the construct pairs mentioned (with slashes between the names of two constructs). The vectors pDL2-Nx and pDL2-xN containing no CYB5s were used as negative controls (where x indicates the control Alg5 cDNA). The streaks stained blue indicate the interactions of MdSUT1 with AtCYB5-A (MdSUT1/pDL2-Nx-AtCYB5-A) and with ScCYB5 (MdSUT1/pDL2-Nx-ScCYB5) and those of MdSOT6 with AtCYB5-A (MdSOT6/pDL2-Nx-AtCYB5-A) and with ScCYB5 (MdSOT6/pDL2-Nx-ScCYB5). The yeast cells transformed with the other constructs showed no blue signal. B to D, AtCYB5-A and ScCYB5 coimmunoprecipitate with both MdSUT1 and MdSOT6 in the yeast split-ubiquitin system. The total proteins were prepared from the yeast cells that had been transformed with the construct pairs pTMBV4-MdSUT1/pDL2-Nx-AtCYB5-A (MdSUT1/AtCYB5-A in D), pTMBV4-MdSUT1/pDL2-Nx-ScCYB5 (MdSUT1/ScCYB5 in D), pTMBV4-MdSOT6/pDL2-Nx-AtCYB5-A (MdSOT6/AtCYB5-A in D), and pTMBV4-MdSOT6/pDL2-Nx-ScCYB5 (MdSOT6/ScCYB5 in D). The immunoprecipitates were isolated from the total proteins with the anti-VP16 serum (IP: antiVP16), anti-HA serum (IP: antiHA), anti-MdCYB5 serum (IP: antiCYB5), anti-MdSUT1^CL serum (IP: antiMdSUT1^CL), anti-MdSOT6^CL serum (IP: antiMdSOT6^CL), and the preimmune serum (IP: preimmune serum; as a negative control). The total proteins and immunoprecipitates were immunoblotted with the anti-MdSUT1^CL serum (Blot: anti-MdSUT1^CL; B), anti-MdSOT6^CL serum (Blot: anti-MdSOT6^CL; C), and anti-HA serum (Blot: antiHA; D).

Figure 8.

Low sugar supply stimulates the interactions of MdSUT1 and MdSOT6 with MdCYB5 and AtCYB5-A, and the interactions promote yeast growth in sugar deficiency. A, Low concentrations of Suc stimulate the interactions of MdSUT1 with MdCYB5 and AtCYB5-A (but not ScCYB5). The DSY-1 yeast cells were cotransformed with the vector pairs pTMBV4-MdSUT1 and pDL2-Nx-AtCYB5-A (MdSUT1-AtCYB5-A), pTMBV4-MdSUT1 and pDL2-Nx-MdCYB5 (MdSUT1-MdCYB5), pTMBV4-MdSUT1 and pDL2-Nx-ScCYB5 (MdSUT1-ScCYB5), and pTMBV4-MdSUT1 and pDL2-Nx empty vector (MdSUT1-pDL2-Nx; as a control). The transgenic cells were grown in liquid dropout SD medium lacking Trp and Leu, supplemented with 10 mm Glc and 0.2, 0.5, 1, 2, 4, 8, 12, 20, and 50 mm Suc, respectively, instead of 2% Glc (about 50 mm) supplementation for the common SD medium, and β-gal activities were determined in the lysates of the cells. Relative β-gal activities, normalized relative to the corresponding values obtained from the cells grown at 12 mm Suc, were compared within the same yeast line transformed with the same construct pair. Note that the values of the controls (MdSUT1-pDL2-Nx) were scarcely detectable. The experiments were repeated biologically five times. Values are means ± se (n = 5). B, The interactions of MdSUT1 with MdCYB5 and AtCYB5-A, but not ScCYB5, enhance the yeast growth at low Suc supply. The DSY-1 yeast cells were cotransformed with the vector pairs as described in A. The symbols of the vectors are also the same as in A except for the vector pair pTMBV4-MdSUT1 and pDL2-Nx empty vector, which is called MdSUT1 here. Drop tests were used to observe the transformed yeast growth on low Suc concentration. Cells of 8 μL were spotted at two concentrations (OD₆₀₀ = 0.01 and 0.05) on the media supplemented with 10 or 0.2 mm Suc as sole carbon source and grown at 30°C for 4 d for observations. The assays were repeated three times with the same results. C, Low concentrations of sorbitol stimulate the interactions of MdSOT6 with MdCYB5 and AtCYB5-A (but not ScCYB5). The DSY-1 yeast cells were cotransformed with the vector pairs pTMBV4-MdSOT6 and pDL2-Nx-AtCYB5-A (MdSOT6-AtCYB5-A), pTMBV4-MdSOT6 and pDL2-Nx-MdCYB5 (MdSOT6-MdCYB5), pTMBV4-MdSOT6 and pDL2-Nx-ScCYB5 (MdSOT6-ScCYB5), and pTMBV4-MdSOT6 and pDL2-Nx empty vector (MdSOT6-pDL2-Nx; as a control). The transgenic cells were grown as described in A but with 0.2, 0.5, 1, 2, 4, 8, 12, 20, and 50 mm sorbitol (instead of Suc) in the SD medium, and β-gal activities were determined in the lysates of the cells. Relative β-gal activities, normalized relative to the corresponding values obtained from the cells grown at 8 mm sorbitol, were compared within the same yeast line transformed with the same construct pair. Note that the values of the controls (MdSOT6-pDL2-Nx) were scarcely detectable. The experiments were repeated biologically five times. Values are means ± se (n = 5). D, The interactions of MdSOT6 with MdCYB5 and AtCYB5-A, but not ScCYB5, enhance the yeast growth at low sorbitol supply. The DSY-1 yeast cells were cotransformed with the vector pairs as described in C. The symbols of the vectors are also the same as in C except for the vector pair pTMBV4-MdSOT6 and pDL2-Nx empty vector, which is called MdSOT6 here. Drop tests were conducted as described in B at 10 or 0.2 mm sorbitol as sole carbon source in SD medium. The assays were repeated three times with the same results.

Figure 9.

Expression of MdCYB5 and AtCYB5-A enhances the affinity of MdSUT1 and MdSOT6 to their substrate sugars. A to C, Expression of MdCYB5 and AtCYB5-A enhances the affinity of MdSUT1 to Suc. SUSY7/ura3 yeast cells were transformed with the pDR196 vector harboring MdSUT1 (indicated by MdSUT1), mutated MdSUT1 at Leu-73→Pro (MdSUT1^L73-P), and MdCYB5 (as a negative control) or with the empty pDR196 vector (as another negative control). The yeast cells were also cotransformed with the pDR196 vector pairs pDR196-MdSUT1 plus pDR196-MdCYB5 (MdSUT1+MdCYB5), pDR196-MdSUT1 plus pDR196-AtCYB5-A (MdSUT1+AtCYB5-A), and pDR196-MdSUT1^L73-P plus pDR196-MdCYB5 (MdSUT1^L73-P+MdCYB5). The time course of Suc uptake by the transgenic yeast cells was assayed for [¹⁴C]Suc uptake at 0.4 mm Suc and pH 5.0 (A). Suc uptake kinetics of the transgenic yeast cells were assayed at pH 5.0 at low Suc concentrations ranging from 0.2 to 2 mm (B). The Suc uptake rates were plotted against the Suc concentrations (C) according to the values presented in B. A linear regression fit of the Michaelis-Menten equation was obtained for each yeast line expressing the mutated MdSUT1^L73-P or wild-type MdSUT1 and CYB5s. The Suc uptake kinetics parameters for these transgenic yeast lines are presented in Table II. Symbols in C are as described in A. D to F, Expression of MdCYB5 and AtCYB5-A enhances the affinity of MdSOT6 to sorbitol. RS453 yeast cells were transformed with the pDR196 vector harboring MdSOT6 (indicated by MdSOT6), mutated MdSOT6 at Leu-117→Pro (MdSOT6^L117-P), and MdCYB5 (as a negative control) or with the empty pDR196 vector (as another negative control). The yeast cells were also cotransformed with the pDR196 vector pairs pDR196-MdSOT6 plus pDR196-MdCYB5 (MdSOT6+MdCYB5), pDR196-MdSOT6 plus pDR196-AtCYB5-A (MdSOT6+AtCYB5-A), and pDR196-MdSOT6^L117-P plus pDR196-MdCYB5 (MdSOT6^L117-P+MdCYB5). The time course of sorbitol uptake by the transgenic yeast cells was assayed for [³H]sorbitol uptake at 0.25 mm sorbitol and pH 4.5 (D). Sorbitol uptake kinetics of the transgenic yeast cells were assayed at pH 4.5 at low sorbitol concentrations ranging from 0.2 to 2 mm (E). The sorbitol uptake rates were plotted against the sorbitol concentrations (F) according to the values presented in E. A linear regression fit of the Michaelis-Menten equation was obtained for each yeast line expressing the mutated MdSOT6^L117-P or wild-type MdSOT6 and CYB5s. The sorbitol uptake kinetics parameters for these transgenic yeast lines are presented in Table II. Symbols in F are as described in D.

Figure 10.

Coexpression of MdSUT1 or MdSOT6 with MdCYB5 or AtCYB5-A promotes yeast growth in sugar deficiency. A, Coexpression of MdSUT1 with MdCYB5 or AtCYB5-A in the SUSY7/ura3 yeast cells. Yeast cells were transformed with the pDR196 vector harboring MdSUT1 (indicated by MdSUT1), mutated MdSUT1 at Leu-73→Pro (MdSUT1^L73-P), and MdCYB5 (as a negative control) or with the empty pDR196 vector (pDR196, as another negative control). The yeast cells were also cotransformed with the pDR196 vector pairs pDR196-MdSUT1 plus pDR196-MdCYB5 (MdSUT1+MdCYB5), pDR196-MdSUT1 plus pDR196-AtCYB5-A (MdSUT1+AtCYB5-A), pDR196-MdSUT1^L73-P plus pDR196-MdCYB5 (MdSUT1^L73-P+MdCYB5), and pDR196-MdSUT1^L73-P plus pDR196-AtCYB5-A (MdSUT1^L73-P+AtCYB5-A). The expression of MdSUT1 was tested with immunoblotting using anti-MdSUT1^CL serum, and the expression of MdCYB5 and AtCYB5-A was tested using anti-MdCYB5^N serum in the yeast lysates. Actin was used as a loading control. Drop tests were used to observe the transformed yeast growth on low Suc concentration. Cells of 8 μL were spotted at two concentrations (OD₆₀₀ = 0.01 and 0.05) on the SD media supplemented with 10 or 0.2 mm Suc as sole carbon source and grown at 30°C for 4 d for observations. The assays were repeated three times with the same results. B, Coexpression of MdSOT6 with MdCYB5 or AtCYB5-A in the RS453 yeast cells. Yeast cells were transformed with the pDR196 vector harboring MdSOT6 (indicated by MdSOT6), mutated MdSOT6 at Leu-117→Pro (MdSOT6^L117-P), and MdCYB5 (as a negative control) or with the empty pDR196 vector (pDR196, as another negative control). The yeast cells were also cotransformed with the pDR196 vector pairs pDR196-MdSOT6 plus pDR196-MdCYB5 (MdSOT6+MdCYB5), pDR196-MdSOT6 plus pDR196-AtCYB5-A (MdSOT6+AtCYB5-A), pDR196-MdSOT6^L117-P plus pDR196-MdCYB5 (MdSOT6^L117-P+MdCYB5), and pDR196-MdSOT6^L117-P plus pDR196-AtCYB5-A (MdSOT6^L117-P+AtCYB5-A). The expression of MdSOT6 was tested with immunoblotting using anti-MdSOT6^CL serum, and the expression of MdCYB5 and AtCYB5-A was tested using anti-MdCYB5^N serum in the yeast lysates. Actin was used as a loading control. Drop tests were used to observe the transformed yeast growth on low sorbitol concentration as described in A, but on the SD media supplemented with 10 or 0.2 mm sorbitol as sole carbon source. The assays were repeated three times with the same results.

Figure 11.

A model of sugar sensing through the MdSUT1-MdCYB5 or MdSOT6-MdCYB5 complex. A, A scheme showing the localization of MdCYB5 to ER. The C-terminal tail is anchored to ER membrane, with the extreme C terminus displayed in the lumen of the ER. The N terminus is present in cytosol and functions to interact with the plasma membrane (PM)-localized sugar transporters MdSUT1 and MdSOT6. B, MdSUT1 (SUT1) or MdSOT6 (SOT6) interacts with MdCYB5 (CYB5) to form an MdSUT1-MdCYB5 or MdSOT6-MdCYB5 bimolecular complex in response to low Suc or sorbitol supply, which enhances the affinity of MdSUT1 or MdSOT6 transporter to its substrate sugar, stimulating sugar uptake by cells to maintain a relatively stable internal level of the limiting sugar. The intensity of the transporter-MdCYB5 interactions and the affinity of the transporter return to normal levels in higher sugar supply.