Immunohistochemical analysis of ZnT1, 4, 5, 6, and 7 in the mouse gastrointestinal tract.

Expression of five zinc transporters (ZnT1, 4, 5, 6, and 7) of the Slc30 family in the mouse gastrointestinal tract was studied by immunohistochemical analysis. Results demonstrated unique expression patterns, levels, and cellular localization among ZnT proteins in the mouse gastrointestinal tract with some overlapping. ZnT1 was abundantly expressed in the epithelium of the esophagus, duodenum of the small intestine, and cecum of the large intestine. ZnT4 was predominantly detected in the large intestine. ZnT5 was mainly expressed in the parietal cell of the stomach and in the absorptive epithelium of the duodenum and jejunum. ZnT6 was predominantly detected in the chief cell of the stomach, columnar epithelial cells of the jejunum, cecum, colon, and rectum. Lastly, ZnT7 was observed in all epithelia of the mouse gastrointestinal tract with the highest expression in the small intestine. Expression of ZnT proteins in the absorptive epithelial cell of the gastrointestinal tract suggests that ZnT proteins may play important roles in zinc absorption and endogenous zinc secretion.     

Functional characterization of a novel mammalian zinc transporter, ZnT6

We describe ZnT6, a new member of the CDF (cation diffusion facilitator) family of heavy metal transporters. The human ZNT6 gene was mapped at 2p21-22, while the mouse Znt6 was localized to chromosome 17. Overexpression of ZnT6 in both wild-type yeast and mutants that are deficient in cytoplasmic zinc causes growth inhibition, but this inhibition is abolished in mutant cells with high cytoplasmic zinc. ZnT6 may function in transporting the cytoplasmic zinc into the Golgi apparatus as well as the vesicular compartment, as evidenced by its overlapping intracellular localization with TGN38 and transferrin receptor in the normal rat kidney cells. We also demonstrate that the intracellular distributions of ZnT6 as well as ZnT4 are regulated by zinc in the normal rat kidney cells. The results from this report, combined with those from other studies, suggest that the intracellular zinc homeostasis is mediated by many ZnT proteins, which act in tissue-, cell-, and organelle-specific manners.     

The ZIP7 gene (Slc39a7) encodes a zinc transporter involved in zinc homeostasis of the Golgi apparatus

It has been suggested that ZIP7 (Ke4, Slc39a7) belongs to the ZIP family of zinc transporters. Transient expression of the V5-tagged human ZIP7 fusion protein in CHO cells led to elevation of the cytoplasmic zinc level. However, the precise function of ZIP7 in cellular zinc homeostasis is not clear. Here we report that the ZIP7 gene is ubiquitously expressed in human and mouse tissues. The endogenous ZIP7 was associated with the Golgi apparatus and was capable of transporting zinc from the Golgi apparatus into the cytoplasm of the cell. Moreover, by using the yeast mutant strain Deltazrt3 that was defective in release of stored zinc from vacuoles, we found that ZIP7 was able to decrease the level of accumulated zinc and in the meantime to increase the nuclear/cytoplasmic labile zinc level in the ZIP7-expressing zrt3 mutant. We showed that the protein expression of ZIP7 was repressed under zinc-rich condition, whereas there were no effects of zinc on ZIP7 gene expression and intracellular localization. Neither did zinc deficiency affect the intracellular distribution of ZIP7 in mammalian cells. Our study demonstrates that ZIP7 is a functional zinc transporter that acts by transporting zinc from the Golgi apparatus to the cytoplasm of the cell.     

Isolation and characterization of Golgi apparatus-specific GODZ with the DHHC zinc finger domain

We identified a novel Golgi apparatus-specific protein with the DHHC zinc finger domain and four putative transmembrane regions, designated as GODZ. The amino acid sequences were highly conserved among mouse and human GODZs and homologous proteins in human, mouse, rat, Drosophila melanogaster, and Caenorhabditis elegans, implying a functional significance of the GODZ protein family. Overexpression of mouse GODZ in COS7 cells suppressed the sorting of the glutamate receptor GluRalpha1 from the Golgi apparatus. These results suggest that GODZ plays a role in the membrane protein trafficking.     

The cytoplasmic tail of alpha 1,2-fucosyltransferase contains a sequence for golgi localization

The Golgi apparatus has a central role in the glycosylation of proteins and lipids. There is a sequential addition of carbohydrates by glycosyltransferases that are distributed within the Golgi in the order in which the glycosylation occurs. The mechanism of glycosyltransferase retention is considered to involve their transmembrane domains and flanking regions, although we have shown that the cytoplasmic tail of alpha1,2-fucosyltransferase is important for its Golgi localization. Here we show that the removal of the alpha1,2-fucosyltransferase cytoplasmic tail altered its function of fucosylation and its localization site. When the tail was removed, the enzyme moved from the Golgi to the trans Golgi network, suggesting that the transmembrane is responsible for retention and that the cytoplasmic tail is responsible for localization. The cytoplasmic tail of alpha1,2-fucosyltransferase contains 8 amino acids (MWVPSRRH), and mutating these to alanine indicated a role for amino acids 3 to 7 in localization with a particular role of Ser(5). Mutagenesis of Ser(5) to amino acids containing an hydroxyl (Tyr and Thr) demonstrated that the hydroxyl at position 5 is important. Thus, the cytoplasmic tail, and especially a single amino acid, has a predominant role in the localization and thus the function of alpha1,2-fucosyltransferase.     

Molecular characterization of KEX1, a kexin-like protease in mouse Pneumocystis carinii

Expression screening of a Pneumocystis carinii-infected mouse lung cDNA library with specific monoclonal antibodies (mAbs) led to the identification of a P. carinii cDNA with extensive homology to subtilisin-like proteases, particularly fungal kexins and mammalian prohormone convertases. The 3.1 kb cDNA contains a single open reading frame encoding 1011 amino acids. Structural similarities to fungal kexins in the deduced primary amino acid sequence include a putative proenzyme domain delineated by a consensus autocatalytic cleavage site (Arg-Glu-Lys-Arg), conserved Asp, His, Asn and Ser residues in the putative catalytic domain, a hydrophobic transmembrane spanning domain, and a carboxy-terminal cytoplasmic domain with a conserved tyrosine motif thought to be important for localization of the protease in the endoplasmic reticulum and/or Golgi apparatus. Based on these structural similarities and the classification of P. carinii as a fungus, the protease was named KEX1. Southern blotting of mouse P. carinii chromosomes localized kex1 to a single chromosome of approximately 610 kb. Southern blotting of restriction enzyme digests of genomic DNA from P. carinii-infected mouse lung demonstrated that kex1 is a single copy gene. The function of kexins in other fungi suggests that KEX1 may be involved in the post-translational processing and maturation of other P. carinii proteins.     

Two different zinc transport complexes of cation diffusion facilitator proteins localized in the secretory pathway operate to activate alkaline phosphatases in vertebrate cells

Zinc is an essential component for the catalytic activity of numerous zinc-requiring enzymes. However, until recently little has been known about the molecules involved in the pathways required for supplying zinc to these enzymes. We showed recently (Suzuki, T., Ishihara, K., Migaki, H., Matsuura, W., Kohda, A., Okumura, K., Nagao, M., Yamaguchi-Iwai, Y., and Kambe, T. (2005) J. Biol. Chem. 280, 637-643) that zinc transporters, ZnT5 and ZnT7, are required for the activation of zinc-requiring enzymes, alkaline phosphatases (ALPs), by transporting zinc into the lumens of the Golgi apparatus and the vesicular compartments where ALPs locate and converting apoALPs to holoALPs. ZnT6 is also located in the vesicular compartments like ZnT5 and ZnT7. However, the functions of ZnT6 and relationships among these three transporters have not been characterized yet. Here, we characterized the cellular function of ZnT6 together with ZnT5 and ZnT7 by gene-targeting studies using DT40 cells. ZnT6-deficient DT40 cells showed low ALP activity, suggesting that ZnT6 is required for the activation of zinc-requiring enzymes like ZnT5 and ZnT7. Combined disruptions of three transporter genes and re-expressions of transgenes revealed that ZnT5 and ZnT6 work in the same pathway, whereas ZnT7 acts alone. Furthermore, co-immunoprecipitation studies revealed that ZnT5 and ZnT6 formed hetero-oligomers, whereas ZnT7 formed homo-oligomers. Interestingly, the Ser-rich loop in ZnT6, a potential zinc-binding site, was dispensable for the zinc-supplying function of ZnT5/ZnT6 hetero-oligomers, suggesting that the His-rich loop in ZnT5 may be important for zinc binding and that the loop in ZnT6 may acquire another function in the hetero-oligomer formation. These results suggest that two different zinc transport complexes operate to activate ALPs.     

ZnT5 variant B is a bidirectional zinc transporter and mediates zinc uptake in human intestinal Caco-2 cells

Zinc is an essential micronutrient, so it is important to elucidate the molecular mechanisms of zinc homeostasis, including the functional properties of zinc transporters. Mammalian zinc transporters are classified in two major families: the SLC30 (ZnT) family and the SLC39 family. The prevailing view is that SLC30 family transporters function to reduce cytosolic zinc concentration, either through efflux across the plasma membrane or through sequestration in intracellular compartments, and that SLC39 family transporters function in the opposite direction to increase cytosolic zinc concentration. We demonstrated that human ZnT5 variant B (ZnT5B (hZTL1)), an isoform expressed at the plasma membrane, operates in both the uptake and the efflux directions when expressed in Xenopus laevis oocytes. We measured increased activity of the zinc-responsive metallothionein 2a (MT2a) promoter when ZnT5b was co-expressed with an MT2a promoter-reporter plasmid construct in human intestinal Caco-2 cells, indicating increased total intracellular zinc concentration. Increased cytoplasmic zinc concentration mediated by ZnT5B, in the absence of effects on intracellular zinc sequestration by the Golgi apparatus or endoplasmic reticulum, was also confirmed by a dramatically enhanced signal from the zinc fluorophore Rhodzin-3 throughout the cytoplasm of Caco-2 cells overexpressing ZnT5B at the plasma membrane when compared with control cells. Our findings demonstrate clearly that, in addition to mediating zinc efflux, ZnT5B at the plasma membrane can function to increase cytoplasmic zinc concentration, thus indicating a need to reevaluate the current paradigm that SLC30 family zinc transporters operate exclusively to decrease cytosolic zinc concentration.     

Zinc transporters, ZnT5 and ZnT7, are required for the activation of alkaline phosphatases, zinc-requiring enzymes that are glycosylphosphatidylinositol-anchored to the cytoplasmic membrane

Numerous proteins are properly folded by binding with zinc during their itinerary in the biosynthetic-secretory pathway. Several transporters have been implicated in the zinc entry into secretory compartments from cytosol, but their precise roles are poorly understood. We report here that two zinc transporters (ZnT5 and ZnT7) localized in the secretory apparatus are responsible for loading zinc to alkaline phosphatases (ALPs) that are glycosylphosphatidylinositol-anchored membrane proteins exposed to the extracellular site. Disruption of the ZnT5 gene in DT40 cells decreased the ALP activity to 45% of that in the wild-type cells. Disruption of the ZnT7 gene lowered the ALP activity only by 20%. Disruption of both genes markedly decreased the ALP activity to <5%. Overexpression of human ZnT5 or ZnT7 in DT40 cells deficient in both ZnT5 and ZnT7 genes recovered the ALP activity to the level comparable to that in the wild-type cells. The inactive ALP protein in DT40 cells deficient in both ZnT5 and ZnT7 genes was transported to cytoplasmic membrane like the active ALP protein in the wild-type cells. Thus both ZnT5 and ZnT7 contribute to the conversion of apo-ALP to holo-ALP.     

Demonstration and Characterization of the Heterodimerization of ZnT5 and ZnT6 in the Early Secretory Pathway

The majority of CDF/ZnT zinc transporters form homo-oligomers. However, ZnT5, ZnT6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. The details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other CDF/ZnT family proteins. Here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, and chimera studies of hZnT5 and hZnT6 in chicken DT40 cells deficient in ZnT5, ZnT6, and ZnT7 proteins. We found that hZnT5 and hZnT6 combine to form heterodimers but do not form complexes larger than heterodimers. Mutagenesis of hZnT6 indicated that the sites present in transmembrane domains II and V in which many CDF/ZnT proteins have conserved hydrophilic amino acid residues are not involved in zinc binding of hZnT6, although they are required for zinc transport in other CDF/ZnT family homo-oligomers. We also found that the long N-terminal half of hZnT5 is not necessary for its functional interaction with hZnT6, whereas the cytosolic C-terminal tail of hZnT5 is important in determining hZnT6 as a partner molecule for heterodimer formation. In DT40 cells, cZnT5 variant lacking the N-terminal half was endogenously induced during periods of endoplasmic reticulum stress and so seemed to function to supply zinc to zinc-requiring enzymes under these conditions. The results outlined here provide new information about the mechanism of action through heterodimerization of CDF/ZnT proteins that function in the early secretory pathway.     

Znt7 (Slc30a7)-deficient mice display reduced body zinc status and body fat accumulation

In vitro studies have demonstrated that ZNT7 is involved in transporting the cytoplasmic zinc into the Golgi apparatus of the cell for zinc storage or to be incorporated into newly synthesized zinc-requiring enzymes/proteins. To evaluate the physiological role of ZNT7, we created a mouse model of Znt7 deficiency by a gene-trap approach. Znt7-deficient mice were zinc-deficient based on their low zinc content in serum, liver, bone, kidney, and small intestine. In embryonic fibroblasts isolated from Znt7-deficient mice, cellular zinc was approximately 50% that of wild-type controls. Znt7-deficient mice also displayed some classic manifestations of dietary zinc deficiency, such as reduced food intake and poor body weight gain. However, the mutant mice did not show any sign of hair abnormality and dermatitis that are commonly associated with dietary zinc deficiency. A radioactive feeding study suggested that Znt7-deficient mice had reduced zinc absorption in the gut resulting in decreased zinc accumulations in other organs in the body. The poor growth found in Znt7-deficient mice could not be corrected by feeding the mutant mice with a diet containing 6-fold higher zinc (180 mg/kg) than the suggested adequate intake amount (30 mg/kg). Furthermore, the reduced body weight gain of the mutant mice was largely due to the decrease in body fat accumulation. We conclude that ZNT7 has essential functions in dietary zinc absorption and in regulation of body adiposity.     

AKAP350 at the Golgi apparatus. I. Identification of a distinct Golgi apparatus targeting motif in AKAP350

The protein kinase A-anchoring proteins (AKAPs) are defined by their ability to scaffold protein kinase A to specific subcellular compartments. Each of the AKAP family members utilizes unique targeting domains specific for a particular subcellular compartment. AKAP350 is a multiply spliced AKAP family member localized to the centrosome and the Golgi apparatus. Three splicing events in the carboxyl terminus of AKAP350 generate the AKAP350A, AKAP350B, and AKAP350C proteins. A monoclonal antibody recognizing all three splice variants as well as a polyclonal antibody specific for AKAP350A demonstrated both centrosomal and Golgi apparatus staining in paraformaldehyde-fixed HCA-7 cells. Golgi apparatus-associated AKAP350A staining was dispersed following brefeldin A treatment. Using GFP chimeric constructs of the carboxyl-terminal regions of AKAP350A, a Golgi apparatus targeting domain was identified between amino acids 3259 and 3307 of AKAP350A. This domain was functionally distinguishable from the recently described centrosomal targeting domain (PACT domain, amino acids 3308-3324) located adjacent to the Golgi targeting domain. These data definitively establish the specific association of AKAP350A with the Golgi apparatus in HCA-7 cells.     

Genomic organization, expression profile, and characterization of the new protein PRA1 domain family, member 2 (PRAF2)

PRA1 domain family, member 2 (PRAF2) is a new 19 kDa protein with four putative transmembrane (TM) domains. PRAF2 (formerly designated JM4) belongs to a new protein family, which plays a role in the regulation of intracellular protein transport. Recently, PRAF2 was found to interact with the chemokine receptor CCR5. In order to further study the function and regulation of PRAF2, we determined its genomic structure and its protein expression pattern in normal and cancerous human tissues. PRAF2 encodes a 178-residue protein, whose sequence is related to PRAF1 (PRA1/prenylin) and PRAF3 (JWA/GTRAP3-18). The human PRAF2 gene contains three exons separated by two introns and is located on human chromosome Xp11.23. The recombinant PRAF2 protein was readily expressed in Schneider 2 (S2) insect cells, and the native protein was detected in human tissues with strong expression in the brain, small intestine, lung, spleen, and pancreas. The protein was undetectable in tissue of the testes. Strong PRAF2 protein expression was also found in human tumor tissues of the breast, colon, lung, and ovary, with a weaker staining pattern in normal tissues of the same patient. Our studies show for the first time that the CCR5-interacting PRAF2 protein is expressed in several human tissues with a possible function in ER/Golgi transport and vesicular traffic.