Plant Cell Rep (2009) 28:1747–1758 between 3 and 5 pmol/mg of protein (Fig. 1). The 3H-IAA binding was decreased in a dose dependent manner by increasing the amounts of non-radioactive IAA in the incubation medium (Fig. 1). The binding of the 3H-IAA to the microsomal membranes was improved signiﬁcantly (15–18-fold) by dialysing the membrane fraction (data not shown). The potential Km for IAA to its binding sites was determined to range in between 3 and 5 lM (Fig. 1). Detection of Funaria ABPs using anti-ABP1 antibodies In order to identify the ABP1 homologue in Funaria, anti- ABP1 antiserum raised against the puriﬁed corn ABP1 was used. The sodium cholate solubilised protein extracts of moss and corn were separated by SDS-PAGE and Western blots were probed with anti-ABP1 antiserum. In corn, which served as a positive control, the antisera cross- reacted with polypeptides of 22 and 24 kDa, however, no polypeptide of similar molecular mass was detectable in the moss extracts. Strikingly, polypeptides of about 31–33 and 46 kDa were detected in both plants tested (Fig. 2). Furthermore, the 31–33 kDa region appears to contain at least two closely migrating polypeptides (Fig. 3). In order Fig. 1 Microsomal membranes from the moss Funaria hygrometrica were incubated on ice with 83 nM 3H IAA for 30 min in dark followed by the addition of increasing concentrations of non- radioactive IAA (0, 6.25, 12.5, 25, 50 and 100 lM) in six different reaction tubes. The reaction mixture was ﬁltered through cellulose acetate ﬁlters, washed with binding buffer and the radioactivity is determined. The standard deviation of triplicate ﬁlters was plotted as a function of concentration of IAA. The concentration corresponding to *Km is shown by an arrow. The variation between samples by the ﬁlter assay was observed to be \5% 1751 Fig. 2 Sodium cholate solubilised total proteins from moss (lane 1) and corn (lane 2) were resolved in 12% SDS-PAGE, transferred to nitrocellulose ﬁlters and probed with anti-ABP1 antibodies or pre- immune sera at a dilution of 1/1,000 Fig. 3 Cell free extracts from moss (M) and corn (C) were fractionated by differential centrifugation to obtain various pellets (P1–P3) and post-microsomal supernatant (Sup) as described in ‘‘Materials and methods’’. Proteins from the pellets were solubilised with 1% sodium cholate and 15 lg of solubilised proteins from each sample were separated using 12% SDS-PAGE, transferred to nitrocellulose ﬁlters and probed with anti-ABP1 antibodies. A 28 kDa putative ABP1 speciﬁc to P3 sub-cellular fraction of moss is marked by an arrow to understand the localisation of these putative ABPs, we performed sub-cellular fractionation using differential centrifugation and probed the Western blots with anti- ABP1 antibodies (Fig. 3). This fractionation led to a sig- niﬁcant enrichment of the 31–33 kDa polypeptides which were detected exclusively in the microsomal membrane fraction and resolved into two or three isoforms (Fig. 3). In addition, a 28 kDa polypeptide was speciﬁcally detected in the P3 sub-cellular fraction of moss (Fig. 3, marked with an arrow). The particulate fractions of moss (Fig. 4a, b) and corn (Fig. 4c, d) were examined by transmission electron microscopy to ascertain the composition of the P2 (Fig. 4a, c) and P3 (Fig. 4b, d) fractions. In both plant species tested, examined fractions contained membranous vesicles. The vesicles in the P2 sub-cellular fractions were often found to be associated with high density dotted structures, presumably ribosomes (Fig. 4a, c). This also implies that the P2 sub-cellular fractions were enriched with vesicles that arose from the intact or broken ER. 123