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Identification of an inhibitory domain of CTP:phosphocholine cytidylyltransferase
1Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor 48109-0606, USA.
Insights
The membrane-binding site of CTP:phosphocholine cytidylyltransferase is within residues 237-314. This region acts as an inhibitory segment, and its removal activates the enzyme for phosphatidylcholine synthesis.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- CTP:phosphocholine cytidylyltransferase (CCT) is a key enzyme in phosphatidylcholine synthesis.
- The enzyme's activity is regulated by membrane association and phosphorylation.
- The role of specific domains, particularly putative amphipathic helices, in CCT regulation is not fully understood.
Purpose of the Study:
- To investigate the function of the amphipathic helices (residues 236-314) in CCT regulation.
- To identify the membrane-binding site and its role in enzyme activation.
- To determine if the 237-314 region acts as an inhibitory segment.
Main Methods:
- Construction and characterization of CCT truncation mutants (CT314 and CT236).
- Expression of mutants in Chinese hamster ovary cells.
- Assays for enzyme activity in vitro and in vivo.
- Analysis of enzyme localization (soluble vs. membrane-associated).
Main Results:
- The membrane-binding site for CCT was localized to residues 237-314.
- CT314 showed membrane association upon oleate treatment, while CT236 remained soluble.
- CT236 exhibited high catalytic activity independent of lipids, suggesting constitutive activation.
- CT314 required lipids for activity, and CT236-expressing cells showed increased phosphatidylcholine synthesis.
Conclusions:
- Residues 237-314 of CCT contain the membrane-binding site.
- This region acts as an autoinhibitory segment, suppressing catalytic activity.
- Removal of this segment, either by truncation or membrane binding, relieves inhibition and activates the enzyme.
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.