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

Abstract

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 Mg 2؉ 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 HIP14⌬DHHC, diminishes HIP14-mediated Mg 2؉ transport by about 50%. Coexpression of an independent protein acyltransferase, GODZ, with the deleted HIP14⌬DHHC mutant restored Mg 2؉ 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 Mg 2؉ transport. We conclude that the huntingtin interacting protein genes, HIP14 and HIP14L, encode Mg 2؉ transport proteins that are regulated by their innate palmitoyl acyltransferases thus fulfilling the characteristics of "chanzymes."

Molecular identification of ancient and modern mammalian magnesium transporters

“…The evidence that HIP14 and HIP14L are Mg 2ϩ transporters is persuasive (40 (65). In confirmation, Varner et al (157) used peptides that mimic distinct palmitoylation motifs and HPLC to measure in vivo palmitoylation.…”

“…Palmitoyl acyltransferases mediate palmitoylation and trafficking of multiple cellular proteins (28,67,175). It was thus surprising that our microarray analysis indicated that the HIP14L transcript was responsive to Mg 2ϩ (40). However, inasmuch as the secondary structures fit our parameters for a candidate Mg 2ϩ transporter, we further characterized their biological functions.…”

“…1) Steady-state membrane currents were recorded with the two-microelectrode voltageclamp technique, and I-V plots were constructed (42). 2) To identify the major cation contributing to the measured currents, we used epifluorescence microscopy with the Mg 2ϩ -responsive mag-fura 2 fluorescent dye to measure Mg 2ϩ flux into single oocytes (38,40). Oocytes were injected with 50 M mag-fura 2 acid 20 min before experimentation.…”

Magnesium in Man: Implications for Health and Disease

“…For example, the nonimprinted in Prader-Willi/Angelman syndrome (NIPA) family of proteins has been proposed to transport Mg 2ϩ , based on Mg 2ϩ currents in Xenopus laevis oocytes (182), but recent studies indicate that NIPA proteins have a role in bone morphogenetic protein (BMP) signaling (525). Likewise, Huntingtininteracting protein 14 (HIP14) was thought to mediate Mg 2ϩ fluxes at the Golgi membrane (183). Now it has become apparent that its main function consists of palmitoyl acyltransferase activity, specifically involved in the palmitoylation of Huntington (129, 582).…”

Transient Receptor Potential Melastatin 7 (TRPM7) Cation Channels, Magnesium and the Vascular System in Hypertension

“…38- 41 Recently it has been shown by microarray analysis that the NIPA1 and 2 genes, named for 'non-imprinted in Prader-Willi/Angelman', membrane Mg 2+ transporters 1 and 2 (MMgT1 and 2 respectively) and Huntington interacting protein genes, HIP14 and HIP14L also encode a Mg 2+ transporter. 42- 44 Analysis of different forms of human disorders characterized by low serum Mg 2+ concentrations due to defective intestinal absorption and/or renal Mg 2+ wasting led to the identification of a paracellular (between cells) magnesium transporter, paracellin-1 (claudin 16), a member of the claudin family of tight-junction proteins. 45 Three members of the TRPM family, TRPM2, TRPM6, and TRPM7, are set apart from other known ion channels because they harbor enzyme domains in their respective COOH termini and thus represent prototypes of an intriguing new protein family of enzyme-coupled ion channels termed 'chanzyme' (channel plus enzyme).…”