Article Figures & Data
Figures
- Figure 1.
Electron Microscopic Localization of LeSUT4.
(A) Immunogold labeling of the sieve element plasma membrane in a potato petiole transverse section using anti-SUT4 antiserum.
(B) Magnification of the plasma membranes of two adjacent sieve elements (boxed in [A]).
(C) Immunogold labeling of a sieve element plasma membrane of tomato adjacent to a phloem parenchyma cell.
(D) Control with IgG-enriched preimmune serum on a transverse section of a sieve element in a tomato petiole.
(E) Magnification of the region boxed in (D) indicating no label of the plasma membrane.
Note the nonspecific cross-reaction of preimmune and immune serum with phloem protein aggregates. cc, companion cells; cw, cell wall; pp, phloem parenchyma cell; se, sieve element.
- Figure 2.
Colocalization of Different Suc Transporters in Sieve Elements on Serial Sections of Potato Source Leaves, Petioles, and Stems.
(A) to (C) Source leaves.
(D) to (F) Petioles.
(G) to (I) Stems.
SUT1 detection ([A], [D], and [G]) was performed using StSUT1-specific antibodies as described previously (Kühn et al., 1997). SUT2 detection ([B], [E], and [H]) and SUT4 detection ([C], [F], and [I]) were performed using LeSUT2 and LeSUT4 antibodies able to recognize their potato orthologs StSUT2 and StSUT4, respectively (Barker et al., 2000; Weise et al., 2000). SUT1 and SUT4 detection was visualized with a fluorescein isothiocyanate–coupled secondary antiserum, and SUT2 detection was visualized with a cy3-coupled secondary antiserum that was excited either via a broad-spectrum filter set (source leaves and stem longitudinal sections) or via a cy3-specific filter set (petiole cross-sections). The same sieve element in the longitudinal serial section is indicated by asterisks. se, sieve elements; sp, sieve plate; x, xylem. Magnification was ×250. Bars = 20 μm in (A) to (C), 40 μm in (D) to (F), and 100 μm in (G) to (H).
- Figure 3.
Fusion Protein Constructs for the Split-Ubiquitin System.
Interactions of LeSUT2-CubPLV were determined with all of the other proteins shown, and interactions of LeSUT1-CubPLV were determined with the other Suc transporter fusion constructs. For the constructs, open reading frames were amplified by PCR and cloned into the expression vectors in frame with the C-terminal (Cub) or the N-terminal (Nub) half of ubiquitin. Restriction sites important for cloning are indicated.
- Figure 4.
Interaction of Two Suc Transporters, LeSUT1 and LeSUT2, with Different NubG Fusion Proteins.
No interaction with the ubiquitin domain NubG, strong interaction with LeSUT1, LeSUT2, and LeSUT4, no interaction with the control protein KAT1, and weak interaction of LeSUT2 with the related protein STP1 were detected. Positive interaction was visualized as β-galactosidase activity as determined by filter assays.
- Figure 5.
Determination of the Functionality of the NubG Fusion of LeSUT1 and AtSTP1.
(A) The SUSY7/ura3 yeast strain was transformed with either the empty pNubG-X vector or with the same vector containing the full-length LeSUT1 open reading frame. The growth of SUSY7/ura3 on Suc depends on the presence of a functional Suc transporter in the plasma membrane. Transformed cells were tested for growth on medium containing either Glc (nonselective) or Suc (selective) as the sole carbon source.
(B) The 18gas yeast strain was transformed with either the empty pNubG-X vector or with the same vector containing the full-length AtSTP1 open reading frame. The growth of 18gas on Glc depends on the presence of a functional Glc transporter in the plasma membrane. Transformed cells were tested for growth on medium containing either maltose (nonselective) or Glc (selective) as the sole carbon source.
- Figure 6.
Suc Uptake into Yeast Cells (SUSY7/ura3) Coexpressing Different Suc Transporters.
(A) StSUT1 under the control of the ADH1 promoter (112A1NE) and empty vector pDR196.
(B) StSUT1 under the control of the ADH1 promoter and no other plasmid.
(C) StSUT1 under the control of the ADH1 promoter (112A1NE) and the PMA1 promoter (112A1NE).
(D) StSUT1 under the control of the PMA1 promoter (pDR196) and no other plasmid.
(E) StSUT1 under the control of the ADH1 promoter, and LeSUT2 under the control of the PMA1 promoter.
(F) StSUT1 under the control of the PMA1 promoter, and LeSUT2 under the control of the ADH1 promoter.
Values presented are means of three measurements ± se.
- Figure 7.
Targeting of SUT1 to the Plasma Membrane in Yeast (SUSY7/ura3) as Visualized by SUT1-GFP Fusions.
(A) Yeast cells expressing only SUT1-GFP.
(B) Yeast cells coexpressing SUT1-GFP and the empty vector pDR196.
(C) Yeast cells coexpressing SUT1-GFP and SUT1.
(D) Yeast cells coexpressing SUT1-GFP and SUT2.
Tables
- Table 1.
Quantitative β-Galactosidase Assays for the Two Bait Constructs LeSUT1-CubPLV and LeSUT2-CubPLV
β-Galactosidase activity Prey construct LeSUT1-CubPLV LeSUT2-CubPLV LeSUT2 0.29 ± 0.03ab 0.17 ± 0.03a LeSUT4 0.66 ± 0.15ab 0.19 ± 0.02a LeSUT1 1.10 ± 0.23a 0.23 ± 0.04a AtSTP1 0.08 ± 0.01c 0.06 ± 0.03b AtKAT1 0.04 ± 0.02c 0.03 ± 0.01c NubG 0.12 ± 0.03b 0.04 ± 0.01c β-Galactosidase activity is shown as pmol o-nitrophenyl β-d-galactopyranoside·min-1·μg protein. Values are means ±sd of four independent measurements. Different letters indicate significant differences (P < 0.05) within one column, as determined by one-way analysis of variance using SPSS statistical software.
