Dimerization and endocytosis of the sucrose transporter StSUT1 in mature sieve elements

Abstract

The sucrose transporter StSUT1 from Solanum tuberosum was shown to be regulated post-translationally by redox reagents. Its activity is increased at least 10-fold in the presence of oxidizing agents if expressed in yeast. Oxidation has also an effect on plasma membrane targeting and dimerization of the protein. In response to oxidizing agents, StSUT1 is targeted to lipid raft-like microdomains and SUT1 protein is detectable in the detergent resistant membrane fraction of plant plasma membranes. Interestingly, StSUT1 treated with brefeldin A seems to aggregate in endocytic compartments in mature sieve elements.¹ Further analysis of SUT1 targeting will certainly provide more information about the putative involvement of lipid raft-like microdomains in endocytic events. We provide here additional information on the dimerization and endocytosis of the SUT1 protein. The oligomerization of overexpressed SoSUT1 from Spinacia oleracea in transgenic potato plants was analyzed by two-dimensional gel electrophoresis and endocytosis of the StSUT1 protein was confirmed by immunogold labeling.

Immunolocalization of StSUT1 in wild-type potato plants showed the highest expression of StSUT1 in sieve elements.² Antisense plants with phloem-specific inhibition of StSUT1 expression, using the companion cell-specific rolC promoter, showed a strong phenotype including leaf chlorosis and reduced growth and development of sink organs³ indicating that StSUT1 is expressed in companion cells.

Potato plants overexpressing the c-myc-tagged SoSUT1 protein showed no modification of the oligomerization behavior of the endogenous StSUT1 protein (Fig. 1, lower). Immunodetection of the overexpressed SoSUT1-myc protein in BN-PAGE showed only a single band corresponding to the dimeric transporter (Fig. 1, upper). ¹⁴C-sucrose transport measurements in plasma membrane vesicles, isolated from this c-myc-tagged line of SoSUT1 overexpressing plants, showed a more than 4-fold increase of proton motive force (pmf)-dependent sucrose uptake compared to wild-type plasma membrane vesicles.⁴ Both the total rate of sucrose uptake and the deduced pmf-dependent sucrose uptake (indicative of H⁺-dependent sucrose transport) was increased in potato plants overexpressing the myc-tagged SoSUT1 protein.⁴ This correlation between transport activity and dimer formation of SUT1 suggests that SoSUT1 is functionally active in potato plants as a dimer. On the basis of the work presented here, we propose that dimerization of SUT1 does not only occur in potato and tomato, but also in spinach. Homodimerization of SUT1 proteins thus seems to be a general phenomenon in phylogenetically very distantly related dicotyledons such as Solanaceae and Amaranthaceae.

Figure 1.

Analysis of transgenic plants overexpressing the c-myc tagged SUT1 protein from spinach under control of the CaMV 35S promoter by two-dimensional gel electrophoresis. In a first dimension, the solubilized plasma membranes were separated under native conditions by blue native PAGE, in a second dimension separation occurred under denaturing conditions in a SDS-PAGE. Immunodetection was performed with either monoclonal c-mycspecific antibodies labeling the overexpressed SoSUT1 protein (upper part) or alternatively with affinity-purified StSUT1-specific antibodies labeling the endogenous StSUT1 protein (lower part).

According to text books cytoskeletal components are absent in mature sieve elements. However, recent proteomic and transcriptomic approaches with phloem exudates together with immunolocalization experiments revealed the presence of actin, myosin and tubulin in mature sieve elements.^5–8

Existence of endocytosis and vesicular recycling processes in mature sieve elements were shown by immunolocalization of StSUT1 with plant material pretreated with brefeldin A when StSUT1 accumulated within endocytic brefeldin A—induced compartments.¹ Immunogold labeling of vesicle-like structures close to the plasma membrane with SUT1-specific antibodies by electron microscopy supports this observation (Fig. 2). We therefore assume that the cellular requirements for endocytic events are present in phloem sieve elements at their mature state. It is still unclear how the SUT1 protein whose gene is expressed in the companion cells can be targeted into the sieve element plasma membrane. As seen by electron microscopy the SUT1 mRNA was detected preferentially at the orifice of plasmodesmata connecting sieve elements and companion cells.² The phloem mobility of SUT1 mRNA was recently shown by hetereograft experiments and between host plants and the parasitic plant Cuscuta reflexa.⁹ Even though SUT1 mRNA was detected in sieve elements, its translation does not necessarily occur here.

Figure 2.

Immunogold labeling of StSUT1 at the plasma membrane of sieve elements (A). Vesicle formation in mature sieve elements in response to pretreatment of the potato leaf tissue with 10 mM EDTA before fixation and embedding. Vesicle diameter is between 150 and 300 nm. (B). If the potato tissue is pretreated before fixation and embedding with 50 µM brefeldin A for 1 h, intracellular compartments are labeled within the sieve elements using SUT1-specific antibodies (C). SE: sieve elements; CC: companion cells; arrows label gold particles.

Using the companion cell specific promoter of the sucrose transporter AtSUC2, a plasma membrane-anchored fluorescent protein fusion was shown to illuminate the plasma membrane and vesicular-like structures within the neighboring sieve elements in Arabidopsis and tobacco.¹⁰ Since we showed co-localization of the SUT1-GFP protein with an ER-marker and intracellular trafficking of SUT1 by GFP fusion as well as by immunolocalization,¹ we suggest that the SUT1 protein is translated in the companion cells and is targeted via the continuous ER strands representing the desmotubulus of plasmodesmata and connecting the ER ER cisternae of the two neighboring cells.¹¹

Addendum to: Krügel U, Veenhoff LM, Langbein J, Wiederhold E, Liesche J, Friedrich T, Grimm B, Martinoia E, Poolman B, Kühn C. Transport and sorting of the Solanum tuberosum sucrose transporter SUT1 is affected by posttranslational modification. Plant Cell. 2008;20:2497–2513. doi: 10.1105/tpc.108.058271.