Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Alignment of Hxk1 and Related Sequences. The protein sequences of plant hexokinases, Hxk1, HXK1 and HXK2 (Arabidopsis thaliana), Hxk2 (Lycopersicon escutelum), HK1 and HK2 (Solanum tuberosum), and HxK1 (Spinacia oleracea, were aligned. The numbers indicate the amino acid residues. The residues that are conserved between the sequences are boxed in black or light gray based on the degree of conservation. The “membrane anchor” sequence is indicated by a dotted line. The second methionine is indicated by the arrow. The phosphate binding sequences are marked with boxes. The sugar recognition sequence is indicated by the bracket. The hexokinase signature and ATP binding sequences are marked by a single and double line, respectively.
• Supplemental Figure 2 - Expression Analysis of Hxk1. (A) Semiquantitative RT-PCR analyses of Hxk1 expression in different plant organs of N. benthamiana. As a control, the actin mRNA levels were examined. (B) Semiquantitative RT-PCR analyses of Hxk1 expression in response to 10 mM H₂O₂ and heat (55 °C) treatments for the indicated times. As a control, the actin mRNA levels were examined. (C) RNA gel blot analysis of Hxk1 expression in leaves in response to treatment with 300 μM thapsigargin for the indicated times, preceded by a 2 h pretreatment with water (-) or 100 μM cycloheximide (ch). Each lane represents 30 μg of total RNA. As a control, the rRNA levels were examined.
• Supplemental Figure 3 - Purification of the Recombinant Hxk1 and Hxk1ΔN. (A) The Hxk1:His_x6 protein (marked by the arrow) was purified as described in the Methods. M and W indicate the prestained molecular weight markers and the Coomassie-stained proteins in the “wash” fraction, respectively. Eluted proteins were also visualized by Coomassie staining (fractions 1 to 8). (B) Purification of the Hxk1ΔN:His_x6 protein (marked by the arrow) that lacks the N-terminal membrane anchor sequence.