In vitro assembly of apophytochrome and apophytochrome deletion mutants expressed in yeast with phycocyanobilin.

Abstract

Recombinant pea type I phytochrome apoprotein expressed in yeast is shown to assemble in vitro with phycocyanobilin to produce a photoreversible phytochromelike adduct. As an initial investigation of the amino acid sequence requirements for chromophore incorporation, three phyA gene product deletion mutants were produced in yeast.

In vitroformation of a photoreversible adduct of phycocyanobilin and tobacco apophytochrome B

“…Compared to the reported values for phytochrome from different sources, the spectra of the PHYB-phycocyanobilin adduct show a blue shift (tobacco PHYA, 730 nm and 666 nm, Cherry et al, 1992;oat PHYA, 731, 667, 400, 379, isosbestic point 689 nm and oat PHYB 727, 656, isosbestic point 681 nm , Pratt et al, 1991 and; Arabidopsis PHYA 730 nm, 665 nm, Parks et al, 1989; Arabidopsis PHYB 729 nm, 664 nm, Wagner et al, 1991; pea PHYB 724 nm and 666 nm, Abe et al, 1985). The measured absorbance values for the PHYB-phycocyanobilin adduct are, however, very similar to those reported for PHYA-phycocyanobilin adducts (pea PHYA-phycocyanobilin adduct, 650 nm and 709 nm, Deforce et al, 1991 ; oat PHYA-phycocyanobilin adduct, 650 nm and 714 nm, Terry and Lagarias, 1991). Therefore, the hypsochromic shift is probably due to the use of phycocyanobilin which possesses a shorter conjugated structure than phytochromobilin.…”

“…Single colonies of the yeast transformants were grown for 48 h on liquid minimal medium [0.67% (mass/vol.) bacto-yeast nitrogen base without amino acids, 0.1 M glucose, 0.1 mM L-tryptophan, 0.23 mM L-leucine, 0.1 mM adenine sulfate] as described by Deforce et al, 1991. The starting culture was diluted 1 : 20 in complete medium [I% (madvol.)…”

“…The purified plasmid DNA was used to transform Saccharomyces cerevisiae strain KN380 (Morishima et al, 1990;Ito et al, 1983). Transformants were identified by plating on minimal medium (Deforce et al, 1991). Single clones were tested for the expression of tobacco PHYB apoproteins by immunodetection using the mATl antibody (L6pez-Juez et al, 1992).…”

“…Besides the description of its spectroscopical properties, PHYA has also been characterized at the molecular level for its destruction mechanism, via the ubiquitin pathway (Speth et al, 1987;Jabben et al, 1989), and for its chromophore attachment (Li and Lagarias, 1992;Deforce et al, 1991). In contrast to PHYA, the very small amount of PHYB in vivo has so far prevented the molecular characterization of this important photoreceptor.…”

“…Phycobilins do require additional enzymes for the formation of phycobiliproteins (Arciero et al, 1988), whereas the attachment of phytochrome chromophores to the apoprotein is probably autocatalytic . Therefore, photoreversible holophytochrome can be generated by in vitro incubation of phytochrome apoproteins with the chromophores (Deforce et al, 1991;Li and Lagarias, 1992). Moreover, feeding experiments with the long hypocotyl mutants (hyl and hy2) of Arubidopsis thuliuna demonstrated that the phytochrome-phycocyanobilin adduct not only exhibits photoreversibility but also possesses biological activity .…”