Molecular properties of a novel, hydrophilic cation-binding protein associated with the plasma membrane

Abstract

A new type of protein was found in Arabidopsis thaliana, PCaP1, which is rich in glutamate and lysine residues. The protein bound (45)Ca(2+) even in the presence of a high concentration of Mg(2+). Real-time polymerase chain reaction and histochemical analysis of promoter-beta-glucuronidase fusions revealed that PCaP1 was expressed in most organs. The PCaP1 protein was detected immunochemically in these organs. Treatment of Arabidopsis seedlings with Cu(2+), sorbitol, or flagellin oligopeptide enhanced the transcription. On the other hand, other sugars, abscisic acid, gibberellic acid, dehydration, and low temperature had little or no effect on PCaP1 transcript abundance. The transient expression of PCaP1 fused to green fluorescent protein in Arabidopsis cells and the subcellular fractionation of tissue homogenate showed that PCaP1 protein is localized to the plasma membrane, although PCaP1 has no predicted transmembrane domain. PCaP1 was associated with the plasma membrane under natural conditions and was released from the membrane at high concentrations of Ca(2+) or Mg(2+) in vitro. These results suggest that the hydrophilic protein PCaP1 binds Ca(2+) and other cations and is stably associated with the plasma membrane.

MDP25, A Novel Calcium Regulatory Protein, Mediates Hypocotyl Cell Elongation by Destabilizing Cortical Microtubules inArabidopsis

“…The plasma membrane localization of MDP25 was examined using hypocotyl epidermal cells of P MDP25 :MDP25:green fluorescent protein (GFP) transgenic lines ( Figure 1M) or protoplasts isolated from MDP25-GFP transgenic lines stained with FM4-64 dye (see Supplemental Figure 2 online). A fluorescence signal was detected at the plasma membrane but not at other cell membranes, which is consistent with previous reports that used transient expression or anti-MDP25 antibody (Ide et al, 2007;Nagasaki et al, 2008). The cells were then treated with the microtubule-disrupting drug oryzalin (10 mM) for 60 min.…”

“…A previous report showed that Ca 2+ can dissociate MDP25 from the plasma membrane in vitro (Ide et al, 2007). Therefore, calcium may be a key factor in regulating the interaction of MDP25 and cortical microtubules in cells.…”

“…It is reported that two hydrophilic cation binding proteins, Arabidopsis plasma membrane-associated cation binding protein 1 (PCaP1) and PCaP2, stably bind to the plasma membrane and to calcium (Ide et al, 2007;Nagasaki et al, 2008;Kato et al, 2010a). PCaP2 is a microtubule-associated protein previously named MAP18, which binds and regulates microtubules (Wang et al, 2007).…”

Application of Radioisotopes in Biochemical Analyses: Metal Binding Proteins and Metal Transporters

“…Radioisotope 45 Ca is necessary to confirm that the protein(s) of interest can bind calcium. The 45 Ca overlay assay is one convenient method (Campbell et al, 1983;Yuasa & Maeshima, 2002;Ide et al, 2007;Kato et al, 2010). If a purified preparation is available, aliquots of the purified sample are spotted onto a membrane filter such as a PVDF membrane (ca.…”

Respuesta de los frutos cítricos a las bajas temperaturas: estudio mediante micromatrices

“…En CN mostró un perfil prácticamente plano durante las cuatro primeras semanas de almacenamiento en frío y sólo se indujo en el estadio tardío de 56 d. En cambio, en F se indujo desde la primera semana, pero sus niveles de expresión apenas alcanzaron los niveles de las muestra de CN previamente a la exposición al frío. El transcrito de At4g20260 se induce por bajas temperaturas (Kawamura y Uemura, 2003), aunque no se detecta el incremento de proteína (Ide et al, 2007). Hay genes que codifican polipéptidos de membrana plasmática que también se inducen con el frío, como un polipéptido de 19 kDa identificado en suspensiones celulares embrionarias de trigo de invierno (tolerante a las bajas temperaturas) tratadas con ABA en frío (Koike et al, 1997).…”