Phosphorylation of Thr-948 at the C terminus of the plasma membrane H(+)-ATPase creates a binding site for the regulatory 14-3-3 protein.

The plant plasma membrane H(+)-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H(+)-ATPase-14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H(+)-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H(+)-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H(+)-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H(+)-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H(+)-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H(+)-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H(+)-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H(+)-ATPase in vivo. Indeed, replacing Thr-948 in the plant H(+)-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H(+)-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H(+)-ATPase activity in the plant and thus for plant growth.

NADPH diaphorase staining within the developing olfactory bulbs of normal and unilaterally odor-deprived rats.

Littermate rat pups underwent either unilateral surgical occlusion of the right external naris or sham surgery on postnatal day 1. At 10, 20 or 30 days postpartum olfactory bulbs were sectioned and stained using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry. Two types of staining were observed and analyzed. The reaction produced a Golgi-like filling of short-axon cells in both deep and superficial bulb areas. No differences in the number, morphology or distribution of these cells were found either across ages or treatment conditions, indicating that the cells are resistant to the effects of the deprivation paradigm. Large regional variations in glomerular and olfactory nerve layer staining density were also observed at each age, reinforcing notions of functional or structural differences between glomeruli at very early ages.