Efflux function, tissue-specific expression and intracellular trafficking of the Zn transporter ZnT10 indicate roles in adult Zn homeostasis.

Zn is essential to the structure and function of numerous proteins and enzymes so requires tight homeostatic control at both the systemic and cellular level. Two families of Zn transporters - ZIP (SLC39) and ZnT (SLC30) - contribute to Zn homeostasis. There are at least 10 members of the human ZnT family, and the expression profile and regulation of each varies depending on tissue type. Little is known about the role and expression pattern of ZnT10; however in silico data predict restricted expression to foetal tissue. We show a differential expression profile for ZnT10 in adult human tissue by RT-qPCR and detect highest levels of expression in small intestine, liver and brain tissues. We present data revealing the functional activity of ZnT10 to be in the efflux direction. Using a plasmid construct to express ZnT10 with an N-terminal FLAG-epitope tag, we reveal subcellular localisation in a neuroblastoma cell line (SH-SY5Y) to be at the Golgi apparatus under standard conditions of culture, with trafficking to the plasma membrane observed at higher extracellular Zn concentrations. We demonstrate down-regulation by Zn of ZnT10 mRNA levels in cultured intestinal and neuroblastoma cell lines and demonstrate reduced transcription from the ZnT10 promoter at an elevated extracellular Zn concentration. These features of ZnT10 localisation, regulation and function, together with the discovery that ZnT10 is expressed a high levels in brain tissue, indicate that ZnT10 has a role in regulating Zn homeostasis in the brain so may have relevance to the development of neurodegenerative disease.

Differential subcellular localization of the splice variants of the zinc transporter ZnT5 is dictated by the different C-terminal regions.

BACKGROUND: Zinc is emerging as an important intracellular signaling molecule, as well as fulfilling essential structural and catalytic functions through incorporation in a myriad of zinc metalloproteins so it is important to elucidate the molecular mechanisms of zinc homeostasis, including the subcellular localizations of zinc transporters. PRINCIPAL FINDINGS: Two splice variants of the human SLC30A5 Zn transporter gene (ZnT5) have been reported in the literature. These variants differ at their N- and C-terminal regions, corresponding with the use of different 5' and 3' exons. We demonstrate that full length human ZnT5 variant B is a genuine transcript in human intestinal cells and confirm expression of both variant A and variant B in a range of untreated human tissues by splice variant-specific RT-PCR. Using N- or C-terminal GFP or FLAG fusions of both isoforms of ZnT5 we identify that the differential subcellular localization to the Golgi apparatus and ER respectively is a function of their alternative C-terminal sequences. These different C-terminal regions result from the incorporation into the mature transcript of either the whole of exon 14 (variant B) or only the 5' region of exon 14 plus exons 15-17 (variant A). CONCLUSIONS: We thus propose that exons 15 to 17 include a signal that results in trafficking of ZnT5 to the Golgi apparatus and that the 3' end of exon 14 includes a signal that leads to retention in the ER.

SIAH1 targets the alternative splicing factor T-STAR for degradation by the proteasome.

T-STAR is one of three members of the SAM68 family of RNA-binding proteins that have been shown to be involved in various gene expression pathways including the control of pre-mRNA splicing. We employed a two-hybrid screen to identify proteins that interact with human T-STAR. The predominant interacting proteins were the E3 ubiquitin ligases SIAH1 and SIAH2. We found that SIAH1 bound to an octapeptide sequence in T-STAR targeting it for proteasome-dependent degradation. Rodent T-STAR orthologues (also known as etoile or SLM2) were not targeted for degradation by SIAH1. However a double amino acid substitution of mouse T-STAR that mimics the human SIAH1-binding site brought mouse T-STAR under in vivo control of SIAH1. Using a minigene transfection assay for alternative splicing activity we showed that human T-STAR, like its rodent orthologues can influence splice site choice and that human, but not mouse, T-STAR-dependent alternative splicing is modulated by SIAH1. Western blots of protein from purified germ cells indicated that SIAH1 protein expression peaks in meiosis. In mouse, T-STAR is co-expressed with SIAH1 during meiosis but, in humans, T-STAR is only strongly expressed after meiosis. Comparative sequence analysis showed SIAH-mediated proteasomal degradation of T-STAR has evolved in the primate lineage. Collectively these data suggest that SIAH-mediated down regulation of alternative splicing may be an important developmental difference between otherwise highly conserved T-STAR proteins.