No abstract

A cDNA coding for the carotenoid biosynthetic enzyme phytoene synthase was cloned from a Narcissus pseudonarcissus flower cDNA library, and the corresponding protein was overexpressed in insect cells using the baculovirus lipofection system. The full-length overexpressed enzyme exhibited very reduced catalytic activity compared with an overexpressed N-truncated form, with its transit sequence removed by site-directed mutagenesis. The shortened form readily bound quantitatively to lipid bilayers. Although it was active with liposomes prepared from plastid lipids, with phospholipid liposomes it was not, even though association took place. In this latter case, free galactose was capable of substituting for galactolipids, resulting in enzymatic activity. It is concluded that galactolipids are involved in catalytic activity, but do not serve as a membrane anchor. Antibodies raised against the recombinant enzyme made it possible to distinguish between a membrane-bound and a soluble, protein-complexed inactive form of phytoene synthase, present in the chromoplast stroma. These findings and data on phytoene synthase mRNA and protein expression presented here are discussed in terms of a possible regulatory role in color formation during chromoplast (flower) development.

The protein‐coding region of the essential Saccharomyces cerevisiae YPT1 gene coding for a ras‐related, guanine‐nucleotide‐binding protein was exchanged in chromosome VI by the protein‐coding segment of either the mouse ypt1 gene or the v‐Ki‐ras gene, and different chimeric YPT1‐v‐Ki‐ras genes. The mouse ypt1 protein with 71% of identical residues compared with the yeast Ypt1 protein could functionally fully replace its yeast homologue as long as the mouse gene was overexpressed under transcriptional control of the inducible GAL10 promoter. In contrast, neither the viral Ki‐ras nor the hybrid proteins were able to substitute for the loss of YPT1 gene function. This study suggests that different parts of the yeast Ypt1 protein are required for the interaction with cellular targets and that these essential parts are conserved in the mammalian ypt1 protein.

The YPT1 gene of the yeast Saccharomyces cerevisiae codes for a guanine nucleotide‐binding protein which is essential for cell viability. Using as hybridization probe cloned yeast YPT1 gene sequences, we have isolated from cDNA libraries prepared from RNA of mouse F9 and C3H10T1/2 cells several overlapping cDNA clones with identical sequence in the regions of overlap. The cDNAs were derived from a gene, designated ypt1, which codes for a protein of 205 amino acids with 71% homology to the yeast YPT1 gene product. Amino acid sequences typical for guanine nucleotide‐binding proteins and characteristic for ypt proteins are perfectly conserved in the mouse ypt1 protein. Two mRNAs of 1600 and 3200 nucleotides, originating from the mouse ypt1 gene and differing in the length of their 3′‐non‐translated region, were identified in mouse F9 cells and in all mouse tissues examined. A monoclonal antibody specifically recognizing the 23.5‐kd yeast YPT1 protein cross‐reacted with a protein of identical size on protein blots of mouse, rat, pig, bovine and human cell lines.

SummaryA cDNA coding for the carotenoid biosynthetic enzyme phytoene desaturase from Narcissus pseudonarcissus was cloned and the corresponding protein expressed in insect cells using the baculovirus system. Polyclonal antibodies raised against the recombinant protein allowed the detection of soluble and tightly membrane-bound populations of phytoene desaturase in the chromoplasts isolated from petals. The soluble form is enzymatically inactive and a constituent of a larger Hsp 7g-containing protein complex in the stroma, whereas the membrane-bound form is functional. In vitro, the soluble form is able to associate on to/into protein-free liposomal membranes made from chromoplast lipids, thereby gaining activity by binding added flavine adenine dinucleotide (FAD). Once bound to membranes, activated phytoene desaturase works independently of any added FAD, employing membrane-bound electron acceptors. FAD, however, exerts no positive effect on the membrane-association process, its role is confined to enzymatic activation. Although carotenoid accumulation is strongly induced during flower development, only very low concentrations of phytoene desaturase transcripts are detectable, while the corresponding protein accumulates in low, but measurable amounts, appearing in soluble and membrane-bound states. Poet-transcriptional mechanisms contribute significantly to carotenoid accumulation, as do factors determining the enzymatic activity of phytoene desaturase, for example by influencing the redox-state of membrane-bound electron acceptors.