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. 2003 Oct 15;23(28):9385-94.
doi: 10.1523/JNEUROSCI.23-28-09385.2003.

ZNRF proteins constitute a family of presynaptic E3 ubiquitin ligases

Toshiyuki Araki  1 , Jeffrey Milbrandt
Affiliations

ZNRF proteins constitute a family of presynaptic E3 ubiquitin ligases

Toshiyuki Araki et al. J Neurosci. .

Abstract

Protein ubiquitination has been implicated recently in neural development, plasticity, and degeneration. We previously identified ZNRF1/nin283, a protein with a unique, evolutionarily conserved C-terminal domain containing a juxtaposed zinc finger/RING finger combination. Here we describe the identification of a closely related protein, ZNRF2, thus defining a novel family of ZNRF E3 ubiquitin ligases. Both ZNRF1 and ZNRF2 have E3 ubiquitin ligase activity and are highly expressed in the nervous system, particularly during development. In neurons, ZNRF proteins are located in different compartments within the presynaptic terminal: ZNRF1 is associated with synaptic vesicle membranes, whereas ZNRF2 is present in presynaptic plasma membranes. Mutant ZNRF proteins with a disrupted RING finger, a domain necessary for their E3 function, can each inhibit Ca2+-dependent exocytosis in PC12 cells. These data suggest that ZNRF proteins play a role in the establishment and maintenance of neuronal transmission and plasticity via their ubiquitin ligase activity.

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Figures

Figure 5.
Figure 5.
ZNRF proteins are located in the presynaptic region in neurons in vivo. Top left, Adult mouse brain homogenate was sequentially centrifuged as schematically represented. Bottom, Immunoblot analysis to examine localization of ZNRF1, ZNRF2, as well as synaptophysin and PSD95. An equivalent amount of protein from each fractionation step was subjected to SDS-PAGE and immunoblot analysis using antibodies to the indicated proteins. Note that the profiles of ZNRF1 and ZNRF2 resemble those of synaptophysin and PSD95, respectively. ZNRF1 is present in LP2 (enriched for synaptic vesicles), whereas ZNRF2 is found in LP1 (synaptic heavy membrane). A-L, Top right, Both ZNRF1 and ZNRF2 are located in presynaptic terminals at the neuromuscular junction. Sections from adult mouse anterior tibial muscle tissue were immunostained using antibodies to ZNRF1 (A, G) and ZNRF2 (D, J) (visualized by Cy3) and compared with localization of SV2 immunoreactivity (B, H; a presynaptic marker, visualized by Alexa488) and binding site of FITC-conjugated α-bungarotixin (E,K; a postsynaptic marker). C,F,I, and L are merged images. Note that immunoreactivity for both ZNRF1 and ZNRF2 is colocalized with SV2 but not with α-bungarotixin-binding site. Scale bars, 5 μm. M-R, Bottom right, ZNRF1 and ZNRF2 are located in synaptic vesicle and on presynaptic terminal, respectively, in primary cultured hippocampal neurons. For synaptic vesicle localization, primary cultured hippocampal neurons were stained by antibodies against ZNRF1 (M) and synaptophysin (N; a synaptic vesicle marker). For presynaptic terminal localization, neurons transiently transfected with a ZNRF2-EGFP expression plasmid (shown in P) were loaded with FM 4-64 dye (Q; a presynaptic terminal marker). O and R are merged images. Scale bars, 20 μm.
Figure 1.
Figure 1.
ZNRF2 contains a zinc finger-RING finger motif homologous to that of ZNRF1. A, Alignment of human ZNRF1 and ZNRF2 amino acid sequences. Residues conserved between ZNRF1 and ZNRF2 are shaded. Cys and His residues of the zinc finger (boxed; residues 145-166 in ZNRF1 and 160-179 in ZNRF2) and RING finger (boxed; residues 184-224 in ZNRF1 and 199-239 in ZNRF2) are in bold. Arrows indicate sites of introns within the coding regions of ZNRF proteins. The ZNRF2 sequence data are available from GenBank/European Molecular Biology Laboratory/DNA Data Bank of Japan under accession numbers AF513707 and AF513708. B, Conserved exon-intron organization in the ZNRF1 and ZNRF2 genes. Genome sequences of ZNRF1 and ZNRF2 at the three intron-exon junctions within the coding region are shown. Nucleotides of the exon are shown in uppercase. The corresponding amino acids are numbered and shown above the nucleotide sequence.
Figure 2.
Figure 2.
ZNRF2 is highly expressed in the nervous system. A, ZNRF2 mRNA levels in human tissues were determined by qRT-PCR. The expression level was normalized to glyceraldehyde-3-phosphate dehydrogenase expression in each sample and is indicated relative to the expression level in liver. All qRT-PCR reactions were performed in duplicate, and the SD is indicated. B-E, G-O, Immunohistochemical analysis of ZNRF2 (B-E, G-J) and ZNRF1 (L-O) in adult mouse (B-M) and rat (I-O) tissues. Signals in cerebellum (B), hippocampus (C), testis (D), hair follicles (E), gut (G, L), trigeminal ganglion (H, M), normal sciatic nerve (I, N), and sciatic nerve (J, O) 7 d after transection are shown. Arrows denote signals in Purkinje cell layer in B, hair matrix (basal portion of hair follicles) in E, and submucosal enteric ganglia in L. Arrowhead in L denotes myenteric ganglia. F, K, Expression of ZNRF2 (F) and ZNRF1 (K) in P0 mouse was examined using in situ hybridization. White arrows denote intense expression of ZNRF2 (F) and ZNRF1 (K) in the cortical plate. Black arrows and arrowheads denote ZNRF2 expression in the gut and adipose tissue, respectively. Note that the hybridization signal is observed predominantly in brain at P0 for both ZNRF1 and ZNRF2 and that the distribution patterns inthe brain are very similar.
Figure 3.
Figure 3.
ZNRF1 and ZNRF2 are E3 ubiquitin ligases. A, In vitro ubiquitination assays were performed with ZNRF1 (top) or ZNRF2 (bottom) in the absence (Mock) or presence of bacterial lysates containing the indicated E2 proteins. Polyubiquitinated proteins generated by in vitro ubiquitination were detected by immunoblot analysis using anti-ubiquitin (Anti-Ub) antibodies. B, In vitro ubiquitination assays were performed with ZNRF1, E1, bacterial lysate containing E2 (UbcH5C), and ubiquitin or in reactions lacking the indicated component (denoted by Δ). Assay mixtures were incubated for 2 hr at 30°C and separated by SDS-PAGE, and polyubiquitinated proteins were detected by immunoblot analysis using anti-ubiquitin antibodies.
Figure 4.
Figure 4.
ZNRF protein ubiquitin ligase activity requires the RING finger motif. A, Schematic representation of wild-type and mutant ZNRF proteins. B, In vitro ubiquitination assays were performed using the E3 proteins indicated in A. Assay mixtures were separated by SDS-PAGE, and polyubiquitinated proteins were detected by immunoblot analysis using anti-ubiquitin (Anti-Ub) antibodies (top). The ZNRF proteins were produced using in vitro transcription-translation-coupling reactions using [35S]methionine-cysteine mixture. Aliquots of the reaction mixtures were separated by SDS-PAGE and subjected to autoradiography to confirm production of the indicated proteins (bottom).
Figure 6.
Figure 6.
ZNRF proteins are located in the endosome-lysosome compartment by N-myristoylation. A, NIH3T3 cells expressing the ZNRF2-EGFP fusion protein were stained with the indicated organelle-specific markers. Note that the EGFP signal colocalized with Lysotracker, a marker for endosome-lysosomes (bottom). Mitotracker, a mitochondrial marker, did not colocalize with the EGFP signal (top). Merge signifies the merged ZNRF2-EGFP and organelle-specific signals. B, NIH3T3 cells were transfected with wild-type or mutant ZNRF1-EGFP fusion protein expression constructs (schematically represented). Twenty-four hours after transfection, the cells were stained with Lysotracker, an endosome-lysosome-specific dye. Note that wild-type ZNRF1, ZNRF1(1-141), ZNRF2(1-156), and ZNRF1(Src1-6) showed speckled patterns and colocalized well with Lysotracker, but ZNRF1(142-227) and ZNRF1(11-227) were located throughout the entire cell. ZNRF1(1-10) associated with membrane structures within the cell but was not located exclusively in the endosome-lysosome compartment. C, NIH3T3 cells transfected with either ZNRF1 or ZNRF1(11-227)-EGFP expression construct (see schematic figure in B) were metabolically labeled with [3H]myristic acid for 4 hr. The cells were lysed, and ZNRF-EGFP fusion proteins were immunoprecipitated using anti-EGFP antibodies. The immunoprecipitated proteins were separated by SDS-PAGE, and myristoylated proteins were detected by autoradiography. Myristoylated ZNRF1 protein is indicated by an arrow.
Figure 7.
Figure 7.
ZNRF proteins play a role in exocytosis. A, PC12 cells were transfected with either hGH expression plasmid alone (indicated by hGH only) or together with expression plasmids for native and mutated ZNRF proteins [WT, wild type; ZNRF1 Mut, ZNRF1(C184A); ZNRF2 Mut, ZNRF2(C199A)]. Transfected cells were incubated at 37°C for 20 min with physiological saline (indicated by PBS), high K+ solution (56 mm) (indicated by High K), or Ca2+ (4.76 mm) under a permeablized condition (indicated by Ca). The amounts of hGH secreted into the medium versus retained in the cells was measured by ELISA, and hGH release was calculated from four independent experiments performed in duplicate as a percentage of total hGH synthesized. **p < 0.01, indicates significant decrease of secreted hGH. B, PC12 cells were cotransfected with a constant amount of hGH plasmid and a varying amount of the indicated ZNRF expression plasmids. Secretion and measurement of hGH and calculation of secreted portion of hGH were performed as described in A. *p < 0.05 and **p < 0.01, indicate significant decrease of secreted hGH.
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