Nuclear export of the rate-limiting enzyme in phosphatidylcholine synthesis is mediated by its membrane binding domain

Karsten Gehrig, Craig C. Morton and Neale D. Ridgway¹

Departments of Pediatrics, and Biochemistry & Molecular Biology, Atlantic Research Centre, Dalhousie University, Halifax, Nova Scotia, Canada

The online version of this article (available at http://www.jlr.org)contains supplementary data in the form of three figures andtwo videos that are in supplementary Figs. I and III.

This research was supported by Canadian Institutes of HealthResearch Operating Grant MOP-62916. K.G. was the recipient ofan Issac Walton Killiam Graduate Studentship. C.C.M. was therecipient of a Nova Scotia Health Research Foundation Studentship.

Published, JLR Papers in Press, December 20, 2008.

^¹ To whom correspondence should be addressed. e-mail: nridgway{at}dal.ca

CTP:phosphocholine cytidylyltransferase ${alpha}$ (CCT ${alpha}$ ), the rate-limitingenzyme in the CDP-choline pathway for phosphatidylcholine (PtdCho)synthesis, is activated by translocation to nuclear membranes.However, CCT ${alpha}$ is cytoplasmic in cells with increased capacityfor PtdCho synthesis and following acute activation, suggestingthat nuclear export is linked to activation. The objective ofthis study was to identify which CCT ${alpha}$ domains were involved innuclear export in response to the lipid activators farnesol(FOH) and oleate. Imaging of CCT-green fluorescent protein (GFP)mutants expressed in CCT ${alpha}$ -deficient CHO58 cells showed that FOH-mediatedtranslocation to nuclear membranes and export to the cytoplasmrequired the membrane binding amphipathic helix (domain M).Nuclear export was reduced by a mutation that mimics constitutivephosphorylation of the CCT phosphorylation (P) domain. However,domain M alone was sufficient to promote translocation to thenuclear envelope and export of a nuclear-localized GFP constructin FOH- or oleate-treated CHO58 cells. In the context of acuteactivation with lipid mediators, nuclear export of CCT-GFP mutantscorrelated with in vitro activity but not PtdCho synthesis.This study describes a nuclear export pathway that is dependenton membrane interaction of an amphipathic helix, thus linkinglipid-dependent activation to the nuclear/cytoplasmic distributionof CCT ${alpha}$ .

Supplementary key words CTP:phosphocholine cytidylyltransferase ${alpha}$ • amphipathic helix • fatty acid • farnesol

Abbreviations: CCT, cytidine triphosphate:phosphocholine cytidylyltransferase; DAG, diacylglycerol; ER, endoplasmic reticulum; FOH, farnesol; GFP, green fluorescent protein; INM, inner nuclear membrane; NE, nuclear envelope; NES, nuclear export signal; NLS, nuclear localization signal; NPC, nuclear pore complex; NR, nucleoplasmic reticulum; PtdCho, phosphatidylcholine

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