Auxin-binding Protein 1 Does Not Bind Auxin within the Endoplasmic Reticulum Despite This Being the Predominant Subcellular Location for This Hormone Receptor

Abstract

Auxin-binding protein 1 (ABP1) is a unique hormone receptor because it resides primarily in the lumen of the endoplasmic reticulum (ER); however, two lines of evidence presented here suggest that ABP1 does not bind auxin within the endoplasmic reticulum, despite its predominant location there. First, ABP1 cannot be photolabeled in intact cells that have accumulated the auxin and photolabeling reagent 5- [7-3 H]azidoindole-3-acetic acid, indicating either that auxin is excluded from the ER and is not available for photolabeling to ABP1 or that binding conditions within the ER lumen are insufficient for photolabeling. Second, at the pH of the ER lumen, auxin binding to ABP1 is not detectable. The pH estimate of the ER lumen is based on an indirect assay, which indicates that the pH is closer to pH 7 than to the binding optimum of pH 5.5. These results indicate that ABP1 does not bind auxin within the ER and point to a site of action that is post-ER. The effect of auxin on its trafficking from the ER was tested in an animal expression system. ABP1 expressed at high levels in COS7 cells is efficiently retained in the ER lumen and is not secreted even in the presence of 190 M indole-3-acetic acid, an auxin concentration that is 40 times above the K d for indole-3-acetic acid binding to ABP1. Hertel et al. (1972) reported auxin-binding in microsomes isolated from corn coleoptile cells and later designated this activity Site I. Several groups (Löbler and Klä mbt, 1985;Shimomura et al., 1986;Napier et al., 1988) purified the protein responsible for this Site I activity (cf. Table I in Jones (1994)), and it has been shown directly that this protein binds auxin .Several lines of evidence indicate that ABP1 1 in maize is an auxin receptor that acts at the plasma membrane. First, among a series of 45 auxins or similar compounds where binding affinity and growth induction was compared, there is a correlation between K d and pC 50 , except with some of the substituted phenoxypropionic acids . A molecular model based on these data, in conjunction with data on the identification of residues in the binding site, point out that auxin binding to ABP1 involves specific molecular interactions, as expected for a receptor (Edgerton et al., 1994;Brown and Jones, 1994). Second, a synthetic peptide encoding the terminal 13 residues of ABP1 significantly modulate the ion current across the plasma membrane of Vicia faba guard cells (Thiel et al., 1993), while synthetic peptides from other regions of ABP1 do not modulate current activity. This suggests that there is a specific interaction between this domain of ABP1 and a plasma membrane component. The behavior of this ABP1 peptide mimics part of the behavior of auxin in the V. faba protoplast (Blatt and Thiel, 1994). Third, antisera directed against ABP1 blocks auxin-induced polarization of the plasma membrane on tobacco mesophyll protoplasts, indicating that ABP1 or an immunochemically similar protein mediates auxin-regulated ion movement (Barbier-Brygoo et al., 1989, 1991Rü ck et al., 19...

The Cytokinin Receptors of Arabidopsis Are Located Mainly to the Endoplasmic Reticulum

“…S1). It is also consistent with optimal cytokinin binding at neutral pH (Romanov et al, 2006), that is, in conditions close to those found in the cytoplasm and ER (Tian et al, 1995), and the proven activity of the cytokinin receptors in diverse membrane systems, including bacteria and yeast (Inoue et al, 2001;Suzuki et al, 2001a). The functionality of ER-located cytokinin receptors is also supported by different active cytokinin pools in plant cells.…”

“…However, additional experiments were not carried out, and localization to endomembranes was not excluded. Instead, biochemical studies of cytokinin receptors revealed that their maximal hormone-binding activity is close to a neutral or weak alkaline pH (Romanov et al, 2006), which is characteristic for the cytoplasm and the endoplasmic reticulum (ER; Tian et al, 1995) but not the apoplast, leading to the suggestion that cytokinin receptors may function inside the cell (Romanov et al, 2006). We here investigate in more detail the subcellular localization of the three cytokinin receptors of Arabidopsis and present biochemical and cell biological evidence indicating that the cytokinin receptors of Arabidopsis are predominantly localized to the ER.…”

AUXIN BINDING PROTEIN1: The Outsider

“…For some time, auxin was suspected to affect secretion by binding to and masking the KDEL motif, but no experimental evidence for this hypothesis could be found (Tian et al, 1995;Henderson et al, 1997). The only effect of auxin on ABP1 localization reported so far is a clustering of ABP1 at the outer membrane of maize coleoptile protoplasts after auxin treatment (Diekmann et al, 1995).…”

RNS2, a conserved member of the RNase T2 family, is necessary for ribosomal RNA decay in plants

“…The environment in the vacuole is different from that found in the ER. Although the vacuolar pH is acidic, the ER interior is maintained at a neutral pH (17,18). Thus, determination of the pH preference of RNS2 activity may indicate whether its biological function is carried out in the ER or in the vacuole.…”