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J. Biol. Chem., Vol. 283, Issue 48, 33365-33374, November 28, 2008

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Huntingtin-interacting Proteins, HIP14 and HIP14L, Mediate Dual Functions, Palmitoyl Acyltransferase and Mg²⁺ Transport^*

Angela Goytain, Rochelle M. Hines, and Gary A. Quamme, Supported by studentships from CIHR and the Michael Smith Foundation for Health Research¹

From the Departments of Medicine and Psychiatry, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Polyglutamine expansions of huntingtin protein are responsible for the Huntington neurological disorder. HIP14 protein has been shown to interact with huntingtin. HIP14 and a HIP14-like protein, HIP14L, with a 69% similarity reside in the Golgi and possess palmitoyl acyltransferase activity through innate cysteine-rich domains, DHHC. Here, we used microarray analysis to show that reduced extracellular magnesium concentration increases HIP14L mRNA suggesting a role in cellular magnesium metabolism. Because HIP14 was not on the microarray platform, we used real-time reverse transcriptase-PCR to show that HIP14 and HIP14L transcripts were up-regulated 3-fold with low magnesium. Western analysis with a specific HIP14 antibody also showed that endogenous HIP14 protein increased with diminished magnesium. Furthermore, we demonstrate that when expressed in Xenopus oocytes, HIP14 and HIP14L mediate Mg²⁺ uptake that is electrogenic, voltage-dependent, and saturable with Michaelis constants of 0.87 ± 0.02 and 0.74 ± 0.07 mM, respectively. Diminished magnesium leads to an apparent increase in HIP14-green fluorescent protein and HIP14L-green fluorescent fusion proteins in the Golgi complex and subplasma membrane post-Golgi vesicles of transfected epithelial cells. We also show that inhibition of palmitoylation with 2-bromopalmitate, or deletion of the DHHC motif HIP14DHHC, diminishes HIP14-mediated Mg²⁺ transport by about 50%. Coexpression of an independent protein acyltransferase, GODZ, with the deleted HIP14DHHC mutant restored Mg²⁺ transport to values observed with wild-type HIP14. Although we did not directly measure palmitoylation of HIP14 in these studies, the data are consistent with a regulatory role of autopalmitoylation in HIP14-mediated Mg²⁺ transport. We conclude that the huntingtin interacting protein genes, HIP14 and HIP14L, encode Mg²⁺ transport proteins that are regulated by their innate palmitoyl acyltransferases thus fulfilling the characteristics of "chanzymes."

Received for publication, February 22, 2008 , and in revised form, September 11, 2008.

^* This work was supported in part by Canadian Institutes of Health Research (CIHR) Grant MOP-53288 (to G. A. Q.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Vancouver Hospital, Koerner Pavilion, 2211 Wesbrook Mall, Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-7156; Fax: 604-822-7897; E-mail: quamme{at}interchange.ubc.ca.

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