Transport and Sorting of the Solanum tuberosum Sucrose Transporter SUT1 Is Affected by Posttranslational Modification

Abstract

The plant sucrose transporter SUT1 from Solanum tuberosum revealed a dramatic redox-dependent increase in sucrose transport activity when heterologously expressed in Saccharomyces cerevisiae. Plant plasma membrane vesicles do not show any change in proton flux across the plasma membrane in the presence of redox reagents, indicating a SUT1-specific effect of redox reagents. Redox-dependent sucrose transport activity was confirmed electrophysiologically in Xenopus laevis oocytes with SUT1 from maize (Zea mays). Localization studies of green fluorescent protein fusion constructs showed that an oxidative environment increased the targeting of SUT1 to the plasma membrane where the protein concentrates in 200-to 300-nm raft-like microdomains. Using plant plasma membranes, St SUT1 can be detected in the detergent-resistant membrane fraction. Importantly, in yeast and in plants, oxidative reagents induced a shift in the monomer to dimer equilibrium of the St SUT1 protein and increased the fraction of dimer. Biochemical methods confirmed the capacity of SUT1 to form a dimer in plants and yeast cells in a redox-dependent manner. Blue native PAGE, chemical cross-linking, and immunoprecipitation, as well as the analysis of transgenic plants with reduced expression of St SUT1, confirmed the dimerization of St SUT1 and Sl SUT1 (from Solanum lycopersicum) in planta. The ability to form homodimers in plant cells was analyzed by the split yellow fluorescent protein technique in transiently transformed tobacco (Nicotiana tabacum) leaves and protoplasts. Oligomerization seems to be cell type specific since under native-like conditions, a phloem-specific reduction of the dimeric form of the St SUT1 protein was detectable in SUT1 antisense plants, whereas constitutively inhibited antisense plants showed reduction only of the monomeric form. The role of redox control of sucrose transport in plants is discussed.

Expression of Sucrose Transporter cDNAs Specifically in Companion Cells Enhances Phloem Loading and Long-Distance Transport of Sucrose but Leads to an Inhibition of Growth and the Perception of a Phosphate Limitation

“…5). Prior reports argue that SUT proteins can form homodimers and heterodimers when expressed in the same cells, and the ratio of monomers to dimers is influenced by protein abundance (Reinders et al, 2002;Krügel et al, 2008). These putative interactions could impart posttranslational control on SUT activity and lead to an inhibition of phloem loading.…”

“…Research presented here demonstrates that while AtSUC2p is repressed, CoYMVp is up-regulated in the presence of elevated Suc levels and thus can be used to bypass possible endogenous regulation of AtSUC2 expression at the transcriptional level. In addition to transcriptional regulation, there is evidence for posttranslational regulation of SUT activity through protein-protein interactions (Schulze et al, 2003;Krügel et al, 2008Krügel et al, , 2009Fan et al, 2009). To potentially bypass these levels of regulation, SUT genes from each of the four subfamily types (Reinders et al, 2012) were tested for the capacity to SUT OE lines perceive a P deficiency as indicated by rescue of wild-type (WT) levels of growth with P supplements and increased expression of PSI genes.…”

Constitutive and Companion Cell-Specific Overexpression of AVP1, Encoding a Proton-Pumping Pyrophosphatase, Enhances Biomass Accumulation, Phloem Loading, and Long-Distance Transport

“…SUT genes that participate in phloem loading are transcriptionally regulated by Suc concentrations (Vaughn et al, 2002), leaf maturation (Riesmeier et al, 1993;Truernit and Sauer, 1995), diurnal cycles (Kühn et al, 1997), and possibly pressure (Smith and Milburn, 1980;Aloni et al, 1986). Posttranslational control may be mediated by protein sorting and proteinprotein interactions (Krügel et al, 2008;Liesche et al, 2010). Overexpression of SUT genes specifically in CCs to enhance loading and carbon partitioning to sink organs has been proposed as a mechanism to improve plant productivity (Ainsworth and Bush, 2011;Braun et al, 2014).…”

Correction

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Based on new results, we wish to correct specific statements made in Krü gel et al (2008).The electrophysiological characterization of Zm SUT1 activation by oxidized glutathione (GSSG), as shown in Figure 4B of Krü gel et al (2008), was due to a change in pH value caused by the application of redox reagents to the medium. Here, we show that at constant pH, the application of GSSG has no affect on sucrose transport in oocytes ( Figure 9A).

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“…Based on these new findings, the yeast uptake experiments reported in Figure 1 of Krü gel et al (2008) were repeated. Application of GSSG strongly affects the pH value of the broadly used 25 mM NaPO 4 buffer, depending on the GSSG concentration.…”