1750 (PMSF) and homogenised for 20 min at 4 C. The suspen- sion was collected in a 1.5 ml polypropylene tube, vortexed vigorously for 3 min and centrifuged at 18,0009g for 5 min. The pellet was again washed with 400 ll of buffer, mixed thoroughly, vortexed for 5 min and used for photo- afﬁnity labelling. Photo-afﬁnity labelling using 5-azido 7-3H-indole 3-acetic acid Photo-afﬁnity labelling reaction was carried out as descri- bed in Jones and Venis (1989) and Jones et al. (1984), with little modiﬁcations. All steps prior to separation of the proteins on the gel were done in dim safe red light in a dark room as described in Jones et al. (1998). The photo-afﬁnity labelling reaction was performed in a ﬁnal volume of 120 ll. Thirty microlitres of labelling buffer (200 mM citrate pH 5.55, 20 mM MgCl2 and 1% sodium cholate) and 80 ll of protein suspension were pipetted into a 1.5 ml polypropylene tube and kept at 4 C. Azido-IAA (0.33 lM ﬁnal) with or without non-radioactive IAA made in water in a ﬁnal volume of 7 ll was added and incubated for 30 min in dark at 4 C. The mixture was then transferred to a glass cavity slide. The slide was kept on a platform maintained at 4 C, 5 cm below the UV source. The con- tents of the slides were exposed to the UV source (3 9 254 nm and 2 9 330 nm bulbs, Hofer UV trans- illuminator from HOFER Scientiﬁc) for 30 s. The samples were frozen immediately in liquid nitrogen and kept at 20 C for 24 h. Subsequently they were boiled with SDS sample buffer containing 62 mM Tris pH 6.8, 10% glyc- erol, 2% SDS, 0.001% bromophenol blue, 5% b-mercap- toethanol, separated using 12% SDS-polyacrylamide gel electrophoresis (PAGE) and analysed by ﬂuorography. For ﬂuorography, the gels were washed in a mixture of 30% ethanol and 12% acetic acid for 2 h. The solution was discarded and replaced with six volumes of inﬁltration solution (55% acetic acid, 15% ethanol, 30% xylene and 0.5% PPO) and agitated for 1 h at room temperature. Care was taken to ensure that the gels were not sticking to the glass tray. The gels were washed two to three times in de- ionised water, dried and autoradiographed for 20–25 days at -80 C. Plant Cell Rep (2009) 28:1747–1758 according to Jones et al. (1984) was purchased from Dr. Alan M. Jones, North Carolina, Chapel Hill, USA. All other chemicals were obtained from Sigma Chemical Co. Phylogeny of ABP1 A phylogenetic tree of ABP1 proteins was constructed using Neighbour Joining as implemented in quicktree (Howe et al. 2002) using the ScoreDist distance matrix (Sonnhammer and Hollich 2005), 1,000 bootstrap replicates and setting the root at the longest internal branch. It includes the ABP1 sequences of 13 land plants, which are indicated by a ﬁve letter code, as follows: Arath: Arabidopsis thaliana (Genbank: NP_192207), Avesa: Avena sativa (Genbank: BAA25433), Cerpu: C. purpureus (Genbank: AAF37576), Cerri: Ceratopteris richardii, Glyma: Glycine max, Medtr: Medicago truncatula, Orysa: Oryza sativa (Genbank: ABF85613), Phypa: P. patens (Cosmoss: Phypa_151546), Poptr: Populus tremula, Selmo: Selaginella moellendorfﬁi, Sorbi: Sorghum bicolor, Vitvi: Vitis vinifera (Genbank: ACG80594), Zeama: Z. mays (Genbank: NP_001105312). Genbank accession numbers can be found on http://www. ncbi.nlm.nih.gov/, while for the other organisms following URLs were used: ftp://ftp.jgi-psf.org/pub/JGI_data/Glycine_max/ annotation/Glyma0.1b.pep.fa.gz http://www.medicago.org/genome/downloads/Mt2/ 20080103_imgag_proteinMAPPED_NO_OVERLAP.fa http://www.cosmoss.org ftp://ftp.jgi-psf.org/pub/JGI_data/Poplar/annotation/ v1.1/proteins.Poptr1_1.JamboreeModels.fasta.gz http://genome.jgi-psf.org/Selmo1/download/ Selmo1_GeneModels_FilteredModels2_aa.fasta.gz ftp://ftp.jgi-psf.org/pub/JGI_data/Sorghum_bicolor/v1.0/ Sorbi1_GeneModels_Sbi1_4_aa.fasta.gz Multiple sequence alignments of all 14 ABP1 sequences were created using MAFFT L-INSI (Katoh et al. 2005) and curated with Jalview (Clamp et al. 2004). Chemicals 123 Anti-ABP1, anti-KDEL and anti-HDEL antibodies were generous gifts from Dr. Mike A. Venis and Dr. Richard M. Napier, HRI, Warwick, UK. The Avidin–Biotin ampliﬁ- cation kit (ABC kit) was purchased from Vector labora- tories, Burlingame, USA, while the PPO was obtained from Sisco chemical laboratories, India. 5-Azido 7-3H-IAA with [99% radiochemical purity by radiochromatography Results 3H-IAA binding to the microsomal membranes Microsomal membranes from Funaria protonema were isolated and observed to exhibit speciﬁc auxin-binding sites. The speciﬁcally bound IAA was estimated to be