Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Supplemental Figure 1. Deduced amino acid sequence alignment for NtRab11b and selected homologs from other plants, yeast and human. AtRABA1f is the Arabidopsis Rab11 homolog that shares the highest homology with NtRab11b. Tomato LeRab11, Arabidopsis ARA4 (AtRABA5c) and AtRABA4b and pea PsRab11 (PRA2) are plant Rab11 homologs for which localization or functional information has been reported (Kang et al., 2001; Nagano et al., 1995; Preuss et al., 2004; Ueda et al., 1996; Lu et al., 2001). The five domains (G1-G5) with highly conserved amino acid residues play a key role in nucleotide binding and GTP hydrolysis. G1 and G2 are also binding sites for the GTPase activating Proteins. G2 and G3, also known as the switch I and switch II regions of monomeric GTPases are involved in GTP- and Mg²⁺ binding. The highly conserved C-terminal cysteine residues that are involved in membrane attachment are indicated with black diamonds. Arrows at S28 and Q73 indicate where mutations were introduced to create the DN and CA variants of NtRab11b, respectively.
  Acession numbers for the protein sequences compared:
  NtRab11b Acc# mRNA L29269 / protein Q40521 / GI 3024504
  AtRABA1f Acc# gene At5g60860 / protein 10176913
  AtRABA4b Acc# gene At4g39990 / protein 7438426
  AtRABA5c (ARA-4) Acc# gene At2g43130 / protein 114089
  LeRab11a Acc# mRNA AJ245570 /protein CAB65172 / GI 6688534
  PsRab11 (PRA2) Acc# mRNA D12541 / protein BAA02109 / GI 303735
  HsRab11A (H. sapiens) Acc# gene 8766 / protein NP_004654 / GI 4758984
  Ypt31 (S. cerevisiae) Acc# gene 856753/ protein NP_010948 / GI 6320869
• Supplemental Figure 2 - Supplemental Figure 2. Protein blots for the fusion proteins in the pollen from various transgenic plants. (A) Comparable amounts of proteins from Lat52-GFP (line 21) (lane 1), Lat52-GFP-NtRab11b (line 153-12) (lane 2), Lat52-GFP-NtRab11b(DN) (lines 173-4, -8) (lanes 3,4) transformed pollen. (B) Comparable amounts of proteins from Lat52-GFP (lane 1), Lat52-GFP-NtRab11b(CA) (lines 207-2,3,16, 17) (lanes 2-5) transformed pollen. (C) Proteins from Lat52-Sec-GFP, Lat52-SynAGP-GFP and Lat52-NtInv-GFP transformed pollen grains (lanes 1 to 3 respectively). These protein blots were reacted with a polyclonal GFP antibody and an alkaline phosphatase-conjugated secondary antibody (A,C) or a HRP-conjugated secondary antibody followed by chemiluminescent detection(B). The high molecular weight streak of proteins detected in the SynAGP-GFP [lane 2 in (C)] reflects highly glycosylated SynAGP.
• Supplemental Table 1
• Supplemental Movie 1 - Supplemental Movie 1. A confocal time series of an elongating GFP-NtRab11b-expressing pollen tube. Images were taken at 5 sec. intervals. Selected images from this series are shown in Fig. 2.