ZnT-3, a putative transporter of zinc into synaptic vesicles - PubMed (original) (raw)

ZnT-3, a putative transporter of zinc into synaptic vesicles

R D Palmiter et al. Proc Natl Acad Sci U S A. 1996.

Abstract

The murine ZnT3 gene was cloned by virtue of its homology to the ZnT2 gene, which encodes a membrane protein that facilitates sequestration of zinc in endosomal vesicles. ZnT-3 protein is predicted to have six transmembrane domains and shares 52% amino acid identity with ZnT-2, with the homology extending throughout the two sequences. Human ZnT-3 cDNAs were also cloned; the amino acid sequence is 86% identical to murine ZnT-3. The mouse ZnT3 gene has 8 exons and maps to chromosome 5. Northern blot and reverse transcriptase-PCR analyses demonstrate that murine ZnT-3 expression is restricted to the brain and testis. In situ hybridization reveals that within the brain, ZnT-3 mRNA is most abundant in the hippocampus and cerebral cortex. Antibodies raised against the C-terminal tail of mouse ZnT-3 react with the projections from these neurons and produce a pattern similar to that obtained with Timm's reaction, which reveals histochemically reactive zinc within synaptic vesicles. We propose that ZnT-3 facilitates the accumulation of zinc in synaptic vesicles.

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Figures

Figure 1

Figure 1

Comparison of mouse ZnT-3 protein sequence with rat ZnT-2. Amino acids are designated by one-letter code; identical amino acids are connected by lines; similar amino acids are connected by dots; the six predicted transmembrane domains are boxed with Roman numerals.

Figure 2

Figure 2

Sequence of mouse ZnT-3 cDNA, comparison of mouse and human ZnT-3 protein sequences, and mouse ZnT-3 genomic organization. (A) Mouse ZnT-3 cDNA sequence with amino acid sequence in three-letter code above. Differences between human and mouse ZnT-3 are indicated above the mouse protein sequence. Arrows indicate the location of introns and numbers designate the flanking exons. (B) A map of the mouse ZnT-3 genomic DNA showing the location of 8 exons. The regions that have been sequenced are indicated with a dotted line.

Figure 3

Figure 3

Organ distribution of ZnT-3, ZnT-2, and ZnT-1 mRNAs. Total RNA from the indicated organs was prepared and subjected to reverse transcription followed by PCR. The products were electrophoresed through 2.8% agarose and stained with ethidium bromide.

Figure 4

Figure 4

Localization of ZnT-3 mRNA, protein, and histochemically reactive zinc in the brain. Coronal sections of mouse brain were subjected to in situ hybridization with a33P-labeled probe complementary to mouse ZnT-3 mRNA (A and B), immunocytochemistry with affinity-purified antisera raised against the C-terminal tail of ZnT-3 (C and D), and Timm’s staining procedure for vesicular zinc (E and F). (B, D, and F, ×2.8; A, C, and_E_, ×0.7). In the hippocampus, ZnT-3 mRNA is present in the cell bodies of dentate granule cells, and ZnT-3 protein is abundant in the zinc-rich mossy fiber projections emanating from these neurons. A, amygdala (lateral nucleus); H, hippocampus; NCx, neocortex; PCx, piriform cortex; PV, paraventricular thalamic nucleus; ZI, zona inserta; GC, granule cell neurons of the dentate gyrus; hi, hilus; mf, mossy fibers. [Bars = 1000 μm (A) and 200 μm (B).]

Figure 5

Figure 5

Western blot analysis of ZnT-3 protein. Lanes 1–4 represent equivalent amounts of protein isolated from confluent BHK cells: untransfected cells (lane 1), cells expressing ZnT-1 (lane 2), cells expressing ZnT-2 (lane 3), and cells expressing ZnT-3 (lane 4). Lanes 5–7 represent total protein from brain (lane 5), testis (lane 6), and kidney (lane 7). The amount of protein in all lanes was similar by Coomassie blue 250 staining of a replicate gel. Affinity-purified antibody to ZnT-3 was detected with a peroxidase-linked secondary antibody by chemiluminescence.

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