A Second UDP-glucose Pyrophosphorylase Is Required for Differentiation and Development in Dictyostelium discoideum

John D Bishop,Byoung C Moon,Faith Harrow,David Ratner,Richard H Gomer,Robert P Dottin,Derrick T Brazill

doi:10.1074/jbc.m204245200

Abstract

Uridine diphosphoglucose pyrophosphorylase (UDPGP) is a developmentally regulated enzyme in Dictyostelium discoideum, which is involved in trehalose, cellulose, and glycogen synthesis. Two independent UDPGP proteins are believed to be responsible for this activity. To determine the relative contributions of each protein, the genes encoding them were disrupted individually. Cells lacking the udpgp1 gene exhibit normal growth and development and make normal levels of cellulose. In agreement with these phenotypes, udpgp1(-) cells still have UDPGP activity, although at a reduced level. This supports the importance of the second UDPGP gene. This newly identified gene, ugpB, encodes an active UDPGP as determined by complementation in Escherichia coli. When this gene is disrupted, cells undergo aberrant differentiation and development ending with small, gnarled fruiting bodies. These cells also have decreased spore viability and decreased levels of glycogen, whose production requires UDPGP activity. These phenotypes suggest that UgpB constitutes the major UDPGP activity produced during development. Sequence analysis of the two UDPGP genes shows that UgpB has higher homology to other eukaryotic UDPGPs than does UDPGP1. This includes the presence of 5 conserved lysine residues. Udpgp1 only has 1 of these lysines.

Highlights

Uridine diphosphoglucose pyrophosphorylase (UDPGP) is a developmentally regulated enzyme in Dictyostelium discoideum, which is involved in trehalose, cellulose, and glycogen synthesis
We examined the expression of the udpgp1 gene in the disruptant as well as the parental strain by Northern blot analysis of total cytoplasmic RNA
The importance of UDPGP activity has long been implicated in Dictyostelium development [10]

Summary

EXPERIMENTAL PROCEDURES

Growth and Transformation of D. discoideum—D. discoideum strain Ax3 was grown axenically in HL5 medium to a density of 2 ϫ 106 cells/ml or on SM plates in association with Klebsiella bacteria. The assay mixture contained the following: 1 mM UDPGP, 2 nM sodium pyrophosphate, 1.6 mM NADP, 10 ␮M glucose 1,6diphosphate, 1 mM EDTA, 4 mM MgCl2, 0.05 units of phosphoglucomutase, 0.14 units of glucose-6-phosphate dehydrogenase, 85 mM Tricine, pH 7.6, and cell extract to a total volume of 0.5 ml. Glycogen Assay—To determine cellular glycogen content, cells were harvested as vegetative or 20-h developed cultures, washed in PBM, resuspended to 5 ϫ 106 cells/ml in 10% acetic acid, and lysed through a Cameo 25N 5-micron filter (MSI, Westboro, MA). Cells were removed from the filters, washed in PBM, and either serially diluted directly in a Klebsiella aerogenes suspension and plated on SM/5 plates (for cell viability assay), or pelleted, resuspended in 10 mM EDTA/0.1% Nonidet P-40 in PB, incubated at 42 °C for 45 min, washed three times in PBM, and serially diluted in K. aerogenes and plated on SM/5 plates (spore viability assay)

RESULTS

DISCUSSION

Cell line