The retinal rod Na/Ca-K exchanger (NCKX) is a unique calcium extrusion protein utilizing both inward sodium gradient and outward potassium gradient. Three mammalian rod NCKX cDNAs have been cloned to date, but quantitative analysis of NCKX function in heterologous systems has proven difficult. Here, we describe a simple system for quantitative analysis of NCKX function; stable transformation of cultured insect cells with the novel pEA1/153A vector containing NCKX cDNAs was combined with measurements of potassium-dependent 45 Ca uptake in sodium-loaded cells. We carried out structure-function studies on NCKX with the following results: 1) two-thirds of the full-length sequence of bovine NCKX could be deleted without affecting potassiumdependent calcium transport and without affecting key properties of the potassium binding site; 2) the affinity of NCKX for potassium was about 10-fold greater in choline medium when compared with lithium medium; this shift was observed in rod outer segments or in cells expressing full-length rod NCKX, the above deletion mutant, or a distantly related NCKX paralog cloned from Caenorhabditis elegans. We conclude that the potassium binding site is highly conserved among members of the NCKX family and is formed by residues located within the two sets of transmembrane spanning segments in the NCKX sequence.Calcium extrusion across the plasma membrane of cells is vital to all cells, in view of the ubiquitous role of calcium as second messenger and since sustained elevated calcium levels rapidly lead to cell death (1). Calcium extrusion against a large electrochemical calcium gradient is mediated by two classes of plasma membrane proteins, an ATP-driven calcium pump and Na/Ca exchangers. Two groups of plasma membrane Na/Ca exchangers can be distinguished: those that neither require nor transport potassium (the NCX family) and those that require and, in the case of the rod photoreceptor NCKX1, have been demonstrated to transport potassium (the NCKX family) (for recent reviews, see Refs. 2 and 3). To date, three NCKX1 cDNAs have been cloned from mammalian rod photoreceptors (4 -6) and one NCKX2 cDNA from rat brain (7). Furthermore, several potential NCKX paralogs present in lower organisms have been identified on the basis of analysis of sequences obtained from genomic sequencing projects (2, 8). Studies on functional properties of the "in situ" Na/Ca-K exchanger have been limited thus far to NCKX1 found in the plasma membrane of the outer segments of retinal rod photoreceptors (reviewed in Refs. 9 -11). Sequence comparison of the three mammalian NCKX1 orthologs cloned to date reveals a remarkably low sequence identity (ϳ65%) in contrast to sequence identities of Ͼ90% observed for other sodium-coupled transporters. We examined functional activity of heterologously expressed dolphin, bovine, and human NCKX1 in several cell systems and only observed consistent and robust functional expression with the dolphin NCKX1 cDNA (6). Comparing the mammalian rod NCKX1 sequences with the sequence fro...

The ultimate step in the formation of thebaine, a pentacyclic opiate alkaloid readily converted to the narcotic analgesics codeine and morphine in the opium poppy, has long been presumed to be a spontaneous reaction. We have detected and purified a novel enzyme from opium poppy latex that is capable of the efficient formation of thebaine from (7S)-salutaridinol 7-O-acetate at the expense of labile hydroxylated byproducts, which are preferentially produced by spontaneous allylic elimination. Remarkably, thebaine synthase (THS), a member of the pathogenesis-related 10 protein (PR10) superfamily, is encoded within a novel gene cluster in the opium poppy genome that also includes genes encoding the four biosynthetic enzymes immediately upstream. THS is a missing component that is crucial to the development of fermentation-based opiate production and dramatically improves thebaine yield in engineered yeast.

Genes in plant secondary metabolic pathways enable biosynthesis of a range of medically and industrially important compounds, and are often clustered on chromosomes. Here, we study genomic clustering in the benzylisoquinoline alkaloid (BIA) pathway in opium poppy (Papaver somniferum), exploring relationships between gene expression, copy number variation, and metabolite production. We use Hi-C to improve the existing draft genome assembly, yielding chromosome-scale scaffolds that include 35 previously unanchored BIA genes. We find that co-expression of BIA genes increases within clusters and identify candidates with unknown function based on clustering and covariation in expression and alkaloid production. Copy number variation in critical BIA genes correlates with stark differences in alkaloid production, linking noscapine production with an 11-gene deletion, and increased thebaine/decreased morphine production with deletion of a T6ODM cluster. Our results show that the opium poppy genome is still dynamically evolving in ways that contribute to medically and industrially important phenotypes.

The kinetics of symplastic transport in staminal hairs of Setcreasea purpurea was studied. The tip cell of a staminal hair was microinjected with carboxyfluorescein (CF) and the symplastic transport of this CF was videotaped and the digital data analyzed to produce kinetic curves. Using a finite difference equation for diffusion between cells and for loss of dye into the vacuole, kinetic curves were calculated and fitted to the observed data. These curves were matched with data from actual microinjection experiments by adjusting K (the coefficient of intercellular junction diffusion) and L (the coefficient of intracellular loss) until a minimum in the least squares difference between the curves was obtained. (a) Symplastic transport of CF was govemed by diffusion through intercellular pores (plasmodesmata) and intracellular loss. Diffusion within the cell cytoplasm was never limiting. (b) Each cell and its plasmodesmata must be considered as its own diffusion system. Therefore, a diffusion coefficient cannot be calculated for an entire chain of cells. (c) The movement through plasmodesmata in either direction was the same since the data are fit by a diffusion equation. (d) Diffusion through the intercellular pores was estimated to be slower than diffusion through similar pores filled with water.Symplastic transport, the intercellular passage of ions and small molecules in the cytoplasm via plasmodesmata, is required for the proper growth and development of plants (5). The kinetics of symplastic transport is of interest to plant physiologists and has been studied by other researchers. It was proposed ( 14) that: the plasmodesmata constitute the pathway of least resistance for the diffusion of all small solutes; diffusion is the predominant mechanism of transport across the pores for small solutes; solute distribution within the bulk cytoplasm of each cell ought to be by a combination of diffusion and cyclosis; and, there ought to be nearly perfect mixing within the bulk cytoplasm of each cell. Thus, the ratecontrolling factor of symplastic transport should be diffusion through plasmodesmata. When fluorescein entered the symplast through a cut in Tradescantia staminal hairs (15)