Identification of an Inhibitory Domain of CTP:Phosphocholine Cytidylyltransferase

Abstract

The function of the putative amphipathic helices between residues 236 and 314 of CTP:phosphocholine cytidylyltransferase was examined by constructing two truncation mutants; CT314 was missing the entire phosphorylation segment, whereas CT236 was missing both the region with the putative amphipathic helices and the phosphorylation segment. Stable cells lines expressing these truncation mutants in Chinese hamster ovary 58 cells were isolated and characterized. CT314 was predominantly soluble in control cells but became membrane-associated in cells treated with oleate, which also causes translocation of wild-type cytidylyltransferase. CT236 was found to be soluble both in control cells and in cells treated to cause translocation. These results strongly suggest that the membrane-binding site is located within residues 237-314. When assayed for activity in vitro, the mutant forms were catalytically active in the presence of exogenous lipids. CT236, moreover, was as active in the absence of lipids as in their presence, whereas CT314 required lipids for activity. The rate of phosphatidylcholine synthesis in cells expressing CT236 was considerably higher than in wild-type cells, consistent with the enzyme being constitutively active in the cells. These results indicate that residues 237-314 constitute an inhibitory segment; when this segment is removed from the catalytic domain by truncation or by binding to membranes, an inhibitory constraint is removed and cytidylyltransferase is activated.

Enzymatic and Cellular Characterization of a Catalytic Fragment of CTP:Phosphocholine Cytidylyltransferase α

“…CHO58 cells expressing full-length CCT␣ or CCT␣236 were obtained previously (5). Cell stocks were maintained at 34°C in 5% CO 2 in Ham's F-12 medium supplemented with 25 mM HEPES and10% fetal bovine serum (FBS).…”

“…Truncated forms of rat CCT␣ have been constructed to determine the regions responsible for lipid activation. Both full-length CCT␣ and truncated forms lacking the phosphorylation segment require lipids for full activity (4,5). Truncations lacking the M/L region, however, are not lipidactivated (4 -6) and do not translate to membranes in the cell (5).…”

“…Both full-length CCT␣ and truncated forms lacking the phosphorylation segment require lipids for full activity (4,5). Truncations lacking the M/L region, however, are not lipidactivated (4 -6) and do not translate to membranes in the cell (5). It is notable that CHO58 cells that are temperature-sensitive for growth and CCT␣ activity can be complemented by exogenous expression of the truncated CCT␣ lacking the M/L region, which suggested that this segment is not important for growth under normal cell culture conditions (5).…”

“…Thus the precise role of the M/L segment in enzyme activation is not clear. Wang and Kent (5) proposed that the M/L segment interacts with the catalytic domain to cause inhibition and that removal of the M/L segment by lipid binding results in activation of catalysis. Jackowski and coworkers (6), however, proposed that lipids and the M/L domain are co-activators of the catalytic domain.…”

Disruption of CCTβ2 Expression Leads to Gonadal Dysfunction

“…A phospholipid sensor domain, or helical domain (residues 256 to 288), is unique to the mammalian proteins and consists of three consecutive 11-residue repeats that form an amphipathic ␣-helix (30). The helical domain regulates the protein's reversible association with biological membranes (2,18,20,30,31) together with its activity (56,64). The curvature elastic stress hypothesis (4) provides a theoretical framework for understanding both the positive and negative functions of specific membrane lipids in CCT regulation via the helical domain (4,19).…”