Kikawada et al. 10.1073/pnas.0702538104.Supporting InformationFiles in this Data Supplement:SI Figure 8SI Figure 9SI Figure 10SI Materials and MethodsSI Figure 8Fig. 8. Structure of the Tret1 gene and protein. (A) Cloning of Tret1 cDNA from P. vanderplanki. PD1202M17f, PD3608G15f, PD3606D10f, PS1205C05f, PD1204L14f, and PD1202F04f were EST clones of P. vanderplanki. Tret1 cDNA has a single ORF from position 121 to 1,632 in a total of 2,337 bp. (B) BLAST-P search showed that translation product of Tret1 presumably has a conserved domain of a sugar transporter. (C) Topological model for the structure of the TRET1 protein. SOSUI analysis indicated that TRET1 could form a 12-transmembrane structure. We predicted the first loop is extracellular because a typical additional site for an N-linked glycosylation is located in that loop at position 73 of TRET1.SI Figure 9Fig. 9. Cellular localization of TRET1 in Xenopus oocytes. Immunohistochemical staining of cross-sections for oocytes expressing TRET1::AcGFP1 fusion protein (A) or injected water as sham (B) was performed using anti-GFP antibody. At the same time, fluorescence analyses of the cross-sections for the oocytes expressing TRET1::AcGFP1 fusion protein (C), AcGFP1 alone (D), or sham (E) were conducted. These images showed that TRET1::AcGFP1 fusion protein is localized in the membrane.SI Figure 10Fig. 10. Time course of trehalose uptake by Xenopus oocytes injected with either Tret1 cRNA or distilled water as sham were incubated in 105 mM trehalose at 15°C. Each value is a mean ± SEM (n = 3). The Tret1 cRNA-injected oocytes took up trehalose in a time-dependent manner for up to 6 h of incubation, whereas uptake by the water-injected oocytes was negligible.SI Materials and MethodsImmunohistochemical Analysis. The oocytes expressing TRET1::AcGFP1 protein or injected water as sham were fixed in 4% paraformaldehyde in PBS (PBS) for 16 h at 5°C, dehydrated through a graded series of alcohols to 100% EtOH and xylene for each 5 min, and then embedded in paraffin (Wako Pure Chemical Industries, Osaka, Japan). Semithin sections at 5 mm were deparaffinized in xylene and rehydrated thorough a graded series of alcohols to PBS at room temperature (RT). After blocking with 0.3% H2O2-MeOH solution for 10 min and 10% BSA in PBS for 30 min at RT, these sections were incubated with 1:100 dilution of anti-GFP antibody [A.v. monoclonal antibody (JL-8); Clontech] for 1 h at RT, and then stained with Histofine Simple Stain MAX PO kit (Nichirei Biosciences, Tokyo, Japan) according to the manufacturer\'s procedure.Fluorescence Microscopy. The fixed oocytes were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Japan, Tokyo, Japan) and frozen at -80°C. The frozen semithin sections at 6 mm were observed by fluorescence microscopy (Olympus IX70, Tokyo, Japan).