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 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 HIP14DeltaDHHC, diminishes HIP14-mediated Mg2+ transport by about 50%. Coexpression of an independent protein acyltransferase, GODZ, with the deleted HIP14DeltaDHHC 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."
Highlights
The functions of HIP14 are beginning to be clarified
Our data indicates that HIP14 and HIP14L proteins mediate Mg2ϩ transport and the transcripts are regulated by magnesium, indicating that they might play a role in control of cellular magnesium homeostasis
HIP14 and HIP14L Are Magnesium-responsive Genes—With the knowledge that differential gene expression is involved with selective control of epithelial cell magnesium conservation, our strategy was to use microarray analysis to identify cDNAs that were up-regulated with low magnesium [12]
Summary
The functions of HIP14 are beginning to be clarified. The HIP14 secondary structure contains five predicted transmembrane domains that is reminiscent of a membrane receptor or transporter and possesses a cytoplasmic DHHC cysteine-rich domain defined by the Asp-His-His-Cys sequence motif [6]. We have shown that Mg2ϩ entry is through specific and regulated magnesium pathways that are regulated by intrinsic mechanisms such that culture of cells in media containing low magnesium results in up-regulation of Mg2ϩ uptake into the cells These data suggest that epithelial cells can sense the environmental magnesium and through transcription- and translation-dependent processes modulate Mg2ϩ transport and maintain magnesium balance. As the predicted secondary structures of HIP14 and HIP14L amino acid sequences conformed to prototypic membrane transporters, the goal of the present study was to see if the encoded HIP14 and HIP14L proteins mediate Mg2ϩ transport. Our data indicates that HIP14 and HIP14L proteins mediate Mg2ϩ transport and the transcripts are regulated by magnesium, indicating that they might play a role in control of cellular magnesium homeostasis. HIP14-mediated Mg2ϩ transport is regulated by autopalmitoylation through its inherent palmitoyl acyltransferase activity making this an unique membrane transport system
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
