Regulation of Synaptophysin Degradation by Mammalian Homologues of Seven in Absentia *

Synaptophysin is an integral membrane protein of synaptic vesicles characterized by four transmembrane domains with both termini facing the cytoplasm. Although synaptophysin has been implicated in neurotransmitter release, and decreased synaptophysin levels have been associated with several neurodegenerative diseases, the molecular mechanism that regulates the degradation of synaptophysin remains unsolved. Using the cytoplasmic C terminus of synaptophysin as bait in a yeast two-hybrid screen, we identified two synaptophysin-binding proteins, Siah-1A and Siah-2, which are rat homologues of Drosophila Seven in Absentia. We demonstrated that Siah-1A and Siah-2 associate with synaptophysin both in vitro and in vivo and defined the binding domains of synaptophysin and Siah that mediate their association. Siah proteins exist in both cytosolic and membrane-associated pools and co-localize with synaptophysin on synaptic vesicles and early endosomes. In addition, Siah proteins interact specifically with the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 and facilitate the ubiquitination of synaptophysin. Furthermore, overexpression of Siah proteins promotes the degradation of synaptophysin via the ubiquitin-proteasome pathway. Our findings indicate that Siah proteins function as E3 ubiquitin-protein ligases to regulate the ubiquitination and degradation of synaptophysin.

Synaptophysin is an abundant integral membrane protein of synaptic vesicles with four transmembrane domains and a unique cytoplasmic tail rich in proline, glycine, and tyrosine (1)(2)(3). A number of studies have implicated a role for synaptophysin in the regulation of neurotransmitter release and synaptic plasticity (4 -12). In addition, recent studies suggest that synaptophysin also participates in the biogenesis and recycling of synaptic vesicles, perhaps via interactions with cholesterol and dynamin (13,14). During development, synaptophysin expression occurs early in neurogenesis and is greatly up-regulated during synaptogenesis (15)(16)(17). Increases in synaptophysin expression have been found to correlate with long-term potentiation, suggesting that the regulation of synaptophysin expression may contribute to the mechanisms underlying learning and memory (18,19). Conversely, aberrant synaptophysin expression has been associated with several neurodegenerative diseases and psychiatric disorders, such as Alzheimer's disease and schizophrenia (20 -24). In some cases, an alteration in the protein level of synaptophysin is observed without a concomitant change at the mRNA level (22), implying that impaired post-translational regulation may contribute to abnormal synaptophysin expression.
Although ample evidence has implicated a role for synaptophysin in neurotransmitter release, and altered synaptophysin expression has been linked to malfunctions of the nervous system, little is known about the mechanisms that regulate synaptophysin expression, particularly at the post-translational level. To identify proteins that regulate synaptophysin expression and/or function, we preformed a yeast two-hybrid screen in rat brain for proteins that interact with synaptophysin. The cytoplasmic tail of synaptophysin was used as bait, because its unique amino acid composition and structure suggests a possible role in protein-protein interaction (1). From this screen, we isolated two synaptophysin-binding proteins, Siah-1A and Siah-2, which are rat homologues of Drosophila Seven in Absentia (Sina). 1 Sina was originally discovered as a RING finger-containing protein that is critically involved in the neuronal development of the R7 photoreceptor cell in Drosophila (25). Sina functions downstream of the tyrosine kinase receptor Sevenless and Ras/ Raf mitogen-activated protein kinase pathway (26 -28). Recent evidence indicates that Sina, together with phyllopod, promotes the ubiquitin/proteasome-dependent degradation of tramtrack, a negative regulator of neuronal differentiation (29,30). In mammals, there are three highly conserved Sina homologues, Siah-1A, Siah-1B, and Siah-2, which are abundantly expressed in the brain as well as other tissues (31)(32)(33)(34). Siah proteins have been reported to interact with two neuronal membrane proteins, DCC (deleted in colorectal cancer) and group 1-metabotropic glutamate receptors (35,36). Although it remains to be determined if Siahs regulate the degradation of metabotropic glutamate receptors, Siahs have been shown to regulate the degradation of DCC via the ubiquitin-proteasome pathway (35). Deletion analysis reveals that the RING finger * This work was supported by a University of North Carolina Junior Faculty Development Award and a Medical Faculty Award (to L.-S. C.) and by National Institutes of Health Grant NS36320 (to L. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM  containing the N-terminal domain of Siah is required for proteolysis whereas the C terminus is involved in binding DCC (37). Together, these studies suggest that Siah proteins may act to regulate neuronal development and function by mediating the ubiquitin-dependent degradation of a number of neuronal target proteins.
In the current study, we have found that Siah proteins regulate the ubiquitination and degradation of synaptophysin by the proteasome pathway. Thus, Siah proteins may play an important role in the turnover of synaptic vesicle proteins as well as in neurodegenerative diseases where synaptophysin expression is altered.

EXPERIMENTAL PROCEDURES
Yeast Two-hybrid Screen and cDNA Cloning-The bait plasmid, pPC97-SYPtail, was constructed by subcloning the cytoplasmic C-terminal tail (amino acids 218 -307) of rat synaptophysin (38) into the pPC97 vector (39,40). For the two-hybrid screen, the yeast strain CG-1945 (CLONTECH) was transformed sequentially with pPC97-SYPtail and a rat hippocampal/cortical two-hybrid cDNA library (40), using the lithium acetate method (41). Positive clones were selected on 3-aminotriazole (5 mM, Sigma Chemical Co.)-containing medium lacking leucine, tryptophan, and histidine and verified with a filter assay for ␤-galactosidase activity. Prey plasmids were then recovered and retransformed into yeast with pPC97-SYPtail or controls to confirm the specificity of the interaction. The N-terminal sequences of rat Siah-1A and Siah-2 were cloned from rat brain Marathon-Ready cDNA using 5Ј-RACE (rapid amplification of cDNA ends, CLONTECH). All cDNAs were then sequenced multiple times on both strands, using an Applied Biosystems 373A DNA sequencer.
In Vitro Binding Assays-GST fusion proteins or GST control were immobilized on glutathione-agarose beads (Sigma) and incubated with rat brain homogenates or purified S-tag proteins for 2 h at 4°C in 50 mM NaH 2 PO 4 (pH 7.2) and 0.05% Triton X-100, as previously described (41). The incubation mixtures were then pelleted and extensively washed with 50 mM NaH 2 PO 4 (pH 7.2), 150 mM NaCl, and 0.05% Triton X-100. Bound proteins were eluted by boiling in 2ϫ Laemmli sample buffer and analyzed by SDS-PAGE and immunoblotting with appropriate antibodies.
Expression Constructs and Transfections-Conventional molecular biological techniques were used to generate the following expression constructs: pcDNA3-synaptophysin and pcDNA3-myc-synaptophysin, which direct the expression of untagged and N-terminally myc-tagged full-length synaptophysin, respectively; pCHA-Siah-1A, which directs the expression of N-terminally HA-tagged full-length Siah-1A; pEGFP-Siah-1A and pEGFP-Siah-2-⌬2, which direct the expression of N-terminally GFP-tagged, full-length Siah-1A, and truncated Siah-2 (amino acids 224 -325), respectively. Cells were transfected using Lipo-fectAMINE (Invitrogen) as described by the manufacturer. In experiments where the transient expression levels of synaptophysin were compared in the presence and absence of HA-Siah-1A, a luciferase reporter, pRSVL, was co-transfected as an internal control for transfection efficiency (41).
Immunoprecipitations-Extracts were prepared from HeLa or CHO cells transiently transfected with indicated plasmids, and immunoprecipitation were preformed as described previously (42), using anti-HA antibody (3F10), anti-myc (9E10.3), anti-synaptophysin antibody (SVP-38), or control IgG. The immunocomplexes were recovered by incubation with protein G-or protein A-Sepharose beads (Sigma). After extensive washes, the immunocomplexes were dissociated by boiling in the Laemmli sample buffer, and analyzed by SDS-PAGE and immunoblotting.
Immunofluorescence Microscopy-PC12 cells were grown on poly-L-lysine-coated glass coverslips and differentiated with nerve growth factor (NGF, 100 ng/ml) for 48 h. CHO cells were transfected with pEGFP-Siah-1A alone or in combination with pcDNA3-synaptophysin. Cells were fixed for 10 min in 3.4% paraformaldehyde in 1ϫ PBS and permeabilized for 30 min in 1ϫ PBS plus 1% bovine serum albumin and 0.1% saponin prior to incubation with antibodies. The primary antibodies to synaptophysin, Siah or EEA1, were applied in 1ϫ PBS plus 1% bovine serum albumin and 0.1% saponin for 1 h. Cells were incubated for 1 h with secondary antibodies conjugated to Texas Red. Coverslips were mounted in the Vectashield mounting medium (Vector Laboratories, Inc.) and then analyzed using a Leica TCS-NT confocal microscope with fluorescein isothiocyanate and Texas Red filters. The images were then processed using Adobe Photoshop 5.0 (Adobe Systems, Inc.).
Proteolysis Inhibitor Treatment of Cells-PC12 cells or HeLa cells expressing Siah-1A and synaptophysin were incubated for 8 h at 37°C with the proteasome inhibitor MG132 (20 M, Calbiochem), the cysteine protease inhibitor E64 (50 M, Sigma), or vehicle (Me 2 SO, final concentration 0.1%). Cells were then lysed, and the protein concentrations of the lysates were determined by BCA protein assay (Pierce). An equal amount of protein from each lysate was then analyzed by SDS-PAGE and immunoblotting. After incubation for 1 h, the radioactive medium was removed by extensive washes with non-radioactive DMEM. Cells were then incubated for chase intervals of 0, 2, 4, 6, 8, 12, 24, and 48 h in nonradioactive DMEM supplemented with 10% fetal bovine serum and 5ϫ the normal concentration of methionine and cysteine. Cells were lysed after appropriate chase time. An equal amount of protein from each lysate was immunoprecipitated using anti-synaptophysin antibody (SVP-38). Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. The level of synaptophysin was quantified with a Phos-phorImager (Molecular Dynamics).
Ubiquitination Assays-CHO cells were transfected with combinations of the following plasmids: pcDNA3-myc3-synaptophysin, pEGFP-Siah-1A, pEGFP-Siah-1A ⌬N, and pCHA-ubiquitin (43). Twenty-four hours after transfection, the cells were incubated for 8 h with 20 M MG132 (Calbiochem). The cells were then lysed, and an equal amount of protein from each lysate was immunoprecipitated using an anti-myc antibody (9E10.3). Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody to detect synaptophysinpolyubiquitin conjugates.

Identification of Rat Siah-1A and Siah-2 as Synaptophysin-
interacting Proteins-To identify proteins that interact with synaptophysin, we used the yeast two-hybrid system to screen a rat hippocampal/cortical cDNA library with the C-terminal 89 amino acids of synaptophysin as bait. Positive clones were rescued and confirmed by retransformation experiments demonstrating that these clones only interact with synaptophysin, but not with unrelated proteins such as SNAP-25 (data not shown). Out of 6.6 ϫ 10 6 yeast transformants screened, we isolated 35 positive clones, of which three clones encode rat Siah-1A, whereas 32 clones encode rat Siah-2 (Fig. 1A). Previously, three murine Siah proteins, Siah-1A, Siah-1B, and Siah-2, have been described (31). However, only two human homologues, Siah-1A and Siah-2, have been identified, which share ϳ78% amino acid identity with each other (31, 33, 34).
Despite efforts by several different groups, no human cDNAs corresponding to murine Siah-1B could be detected (32,33). Similarly, we could not detect rat cDNAs corresponding to murine Siah-1B by 5Ј-RACE and hybridization studies in the two-hybrid library and other rat cDNA libraries nor in the mRNA preparations. Together, these results suggest that Siah-1B may be a mouse-specific splice variant that does not exist in other species. The facts that Siah-1A and Siah-2 are the only positive synaptophysin-interacting clones and that they were isolated multiple times from the yeast two-hybrid screen The conserved region between Siah-1A and Siah-2 is marked. The locations of the synaptophysin-interacting Siah clones isolated from the yeast two-hybrid screen are indicated below the domain structures. The number of times that each clone was independently isolated is indicated on the right. B, amino acid sequence alignment of the rat Siah proteins with the human and Drosophila homologues. Asterisks indicate the conserved cysteine and histidine residues in the RING finger motif, whereas the solid squares indicate the cysteine and histidine residues in the C/H-rich region.
suggest that the detected interaction between Siah proteins and synaptophysin may be physiologically relevant.
Sina was originally identified as a protein that is required for R7 photoreceptor development in Drosophila (25). Data bank searches reveal the presence of Sina homologues in a number of organisms, including human, mouse, Caenorhabditis elegans, Xenopus, and Arabidopsis. The amino acid sequences of Sina/ Siah proteins are extremely well conserved (31,33,34). For example, rat Siah proteins share ϳ99 and 75% amino acid identity with human Siah proteins and Drosophila Sina, respectively (Fig. 1B). Sina/Siah proteins all contain an N-terminal RING finger motif (C 3 HC 4 ) followed by a conserved cysteine/histidine-rich region (C 2 HC 3 H 2 ), which may represent a novel class of Zn 2ϩ -binding motifs (Fig. 1). RING finger motifs are cysteine/histidine-rich, Zn 2ϩ -binding domains that are thought to mediate protein-protein interactions (44,45). Recent studies indicate that RING finger motifs may function in ubiquitination as ubiquitin ligases via interaction with ubiquitinconjugating enzymes (46,47). Deletion analysis reveals that, although the N-terminal RING finger motif is required for proteolysis function, the C-terminal region of Siah proteins may be involved in the interaction with target proteins (37). Interestingly, the positive synaptophysin-interacting clones, C1, C2, and C3, all contain the C-terminal region of Siah proteins (Fig. 1A), further supporting the notion that the Cterminal region of Siah is the target protein-binding domain.
Siah-1A and Siah-2 Interact with Synaptophysin in Vitro and in Vivo-To determine if the synaptophysin-Siah interaction detected in the yeast two-hybrid system actually occurs in vitro, GST-Siah fusion proteins immobilized on glutathione beads were used to affinity-purify ("pull-down") endogenous synaptophysin from rat brain homogenate. As shown in Fig.  2A, GST fusion proteins, containing either full-length Siah-1A or N-terminally truncated forms of Siah-1A and Siah-2, were able to bind endogenous synaptophysin. In contrast, control GST proteins were unable to bind synaptophysin, confirming the specificity of the binding. To further determine if synaptophysin associates with Siah proteins in vivo, co-immunoprecipitation experiments were performed using lysates of CHO cells co-transfected with synaptophysin and HA-tagged Siah-1A or Siah-2. As shown in Fig. 2B, synaptophysin and HA-Siah-1A were co-immunoprecipitated by the anti-HA antibody but not by the IgG control. Similarly, synaptophysin and HA-Siah-2 (C2) were also co-immunoprecipitated (Fig. 2C). These results confirm the specific association of synaptophysin with Siah proteins in mammalian cells.
Identification of Domains Involved in the Siah-Synaptophysin Association-As shown in Fig. 1, Siah-1A and Siah-2 are highly homologous proteins that share a conserved region with 90% amino acid identity (31,33). The three Siah clones isolated from the yeast two-hybrid screen encode either part or complete sequences of the conserved region (Fig. 1A). To further define the specific domain of Siah proteins responsible for the interaction with synaptophysin, we generated a series of GST fusion proteins containing various truncations of the Siah-2conserved region and analyzed their interaction with endogenous synaptophysin from brain extracts (Fig. 3A). Deletion of the RING finger and the cysteine/histidine-rich motif (Siah-2 ⌬1 and Siah-2 ⌬2) of Siah-2 had no effect on its interaction with synaptophysin, indicating that these regions are not involved in binding synaptophysin. Similarly, the C-terminal residues 278 -325 (Siah-2 ⌬3) are dispensable for association with synaptophysin. However, further deletion of amino acids 225-278 (Siah-2 ⌬4) abolished the ability of Siah to interact with synaptophysin, indicating that this region is necessary for binding synaptophysin. Conversely, GST fusion protein containing amino acids 225-278 (Siah-2 ⌬5) is capable of binding synaptophysin (Fig. 3A). These data indicate that the region between residues 225 and 278 is necessary and sufficient for binding synaptophysin.
To further understand the structural requirements underlying the Siah-synaptophysin interaction, we preformed similar deletion analysis of the cytoplasmic tail of synaptophysin to map the Siah-binding site. A series of S-tag synaptophysin deletion mutants were generated and tested for their ability to bind GST-Siah-1A fusion proteins in binding assays in vitro (Fig. 3B). The cytoplasmic tail of synaptophysin contains nine copies of an imperfect pentapeptide repeat, YGP(Q)QG, with unknown function (1,3). Only the recombinant synaptophysin proteins containing the C-terminal three repeats (Syp ⌬4 and Syp ⌬5) are capable of binding Siah-1A. In contrast, the synaptophysin recombinant proteins containing the other repeats (Syp ⌬1 to Syp ⌬3) were unable to bind Siah.

Siah Exists in Both Cytosolic and Membrane-associated Pools and Colocalizes with Synaptophysin in Transfected CHO
Cells-To investigate the intracellular distribution of Siah proteins, we first performed subcellular fractionation experiments to separate the postnuclear supernatant from HA-tagged Siah-1A-transfected CHO cells into cytosol and membrane fractions. Western blot analysis of these fractions revealed that Siah-1A was present in both the cytosol and membrane fraction, although the relative amount of Siah-1A in the membrane fraction was severalfold more than that in the cytosol fraction (Fig.  4E). To examine the nature of Siah-1A association with membranes, the membrane fraction was extracted with 0.1 M

FIG. 2. In vitro and in vivo interaction between synaptophysin and Siah proteins.
A, GST-Siah fusion proteins were immobilized on glutathione-Sepharose beads and incubated with rat brain homogenate (Input). After extensive washes, bound proteins were eluted and analyzed by SDS-PAGE and immunoblotting using a monoclonal antibody against synaptophysin (upper panel). GST-Siah fusion proteins were shown as Ponceau S staining (lower panel). B, co-immunoprecipitation of synaptophysin with Siah-1A from CHO cells. Extracts from CHO cells co-transfected with pcDNA3-synaptophysin and pCHA-Siah-1A were subjected to immunoprecipitation with anti-HA antibody (3F10) or control rat IgG. The immunoprecipitates were analyzed by immunoblotting for synaptophysin (Syp) and HA-Siah-1A. C, co-immunoprecipitation of synaptophysin with Siah-2 from CHO cells. Extracts from cells co-transfected with pcDNA3-synaptophysin and pCHA-Siah-2 (C2) were immunoprecipitated as described in B. The immunoprecipitates were analyzed by immunoblotting for synaptophysin (Syp) and HA-Siah-2. NaHCO 3 at pH 11.5 or 4 M urea (Fig. 4E). Unlike SNAP-23 (a protein bound to membranes via covalently linked palmitic acids), which was resistant to extraction by high pH and urea, a significant percentage of the Siah-1A protein was extracted by these treatments, suggesting that Siah-1A is peripherally associated with membranes via hydrophilic interactions. It is worthy of note that the extraction of Siah-1A seems to be incomplete, because a significant portion of the Siah-1A protein was detected in the pellet after various treatments. Similar incomplete extraction has been observed for many peripherally associated membrane proteins, such as synapsin I, parkin, and rsec8 (48 -50). The Siah protein in the pellet might represent the denatured protein that has pelleted as an insoluble aggregate and/or the Siah protein trapped inside the membrane vesicles during the subcellular fractionation procedure.
We then use immunofluorescence confocal microscopy to ex-amine the subcellular localization of N-terminally HA-or GFPtagged full-length Siah-1A expressed in CHO cells. Both HAand GFP-tagged Siah-1A exhibited a similar punctate staining pattern (Fig. 4, A and C; data not shown), which is consistent with that observed for FLAG-and myc-tagged Sina and Siah-1 proteins expressed in Drosophila and mammalian cells (33,35,37,51). The punctate staining pattern suggests that Siah proteins are present on vesicular structures, although the identity of such structures has not yet been characterized. Simultaneous staining of cells co-expressing synaptophysin and GFP-Siah-1A with anti-synaptophysin antibody revealed a significant overlap in the distribution of these two proteins (Fig. 4, A  and B), providing additional evidence for their association in vivo. Previous studies have shown that, when expressed in non-neuronal cells such as CHO cells, synaptophysin is targeted to early endosomes (52-56). The co-localization of Siah proteins with synaptophysin suggests that Siah proteins may be localized to early endosomes. To examine this possibility, we compared the distribution of Siah-1A with early endosome antigen 1 (EEA1). EEA1, a core component of early endosome docking and fusion machinery, is one of the most specific markers for early endosomes known to date (57,58). The distribution of Siah-1A significantly overlaps with that of EEA1 (Fig. 4, C  and D), suggesting that at least a fraction of Siah proteins are associated with early endosomes in transfected CHO cells.

Colocalization of Endogenous Siah Proteins with Synaptophysin on Synaptic-like Microvesicles and Early Endosomes in PC12
Cells-Because synaptophysin is specifically expressed in neurons and neuroendocrine cells, it would be of particular interest to determine if endogenous Siah proteins co-localize with synaptophysin in these cells. For characterization of endogenous Siah proteins, a chicken anti-Siah antibody was generated against a 14-amino acid peptide sequence of rat Siah-2, and affinity-purified using the immunogen peptide column. The affinity-purified anti-Siah antibody was used in immunofluorescence studies of endogenous Siah protein localization in rat pheochromocytoma PC12 cells. PC12 is a well characterized neuroendocrine cell line that shares many characteristics of sympathetic neurons, such as secretion of neurotransmitters and the response to nerve growth factor (NGF) (59). For immunofluorescence studies, PC12 cells were treated with NGF to induce the formation of neurites, which are enriched in synaptic-like microvesicles but not endosomes (60). Immunostaining of NGF-differentiated PC12 cells using the affinity-purified anti-Siah antibody revealed a punctate staining pattern, which demonstrates that Siah proteins are localized on vesicular structures in the cell body and neuritic processes (Fig. 5, A and  D). No staining was observed when the pre-immune chicken IgY fraction was used or the anti-Siah antibody was omitted (data not shown), confirming that the Siah staining is specific.
To determine whether endogenous Siah proteins co-localize with synaptophysin, we performed double-immunofluorescence experiments to compare the distribution of Siah proteins with synaptophysin in NGF-differentiated PC12 cells (Fig. 5, A-C). The results revealed a substantial overlap between Siah and synaptophysin immunoreactivity, particularly in the neuritic processes, including the tip of neurites. It is well established that synaptophysin is localized to synaptic-like microvesicles and early endosomes in PC12 cells (52, 54 -56, 61). Moreover, previous studies have shown that early endosomes, as detected by transferrin receptor immunoreactivity or internalized transferrin staining, are restricted to the cell body and are virtually absent from neurites in PC12 cells (52,60). Thus, the overlapping distribution of Siah and synaptophysin in neurites indicates that a significant portion of Siah proteins is localized on synaptic-like microvesicles.
Because a fraction of synaptophysin is localized to early endosomes in PC12 cells and our studies revealed an early endosomal localization of Siah proteins in transfected CHO cells (Fig. 4, C and D), we sought to determine if endogenous Siah proteins are also localized to early endosomes in PC12 cells. Consistent with previous studies (52,60), EEA1 immunostaining is concentrated in the cell body and virtually undetectable in neurites of NGF-differentiated PC12 cells (Fig. 5E). Simultaneous staining of the same cells with anti-Siah and anti-EEA1 antibodies revealed that the distribution of Siah proteins overlapped significantly with that of EEA1 (Fig. 5,  D-F), indicating that a fraction of endogenous Siah proteins is associated with early endosomes.
Regulation of Synaptophysin Stability by Siah Proteins-Because Siah proteins have been shown to regulate the degradation of several interacting proteins, we assessed the effect of Siah overexpression on the expression and stability of synaptophysin. HeLa cells were co-transfected with pcDNA3-synaptophysin and either pCHA-Siah-1A or pCHA vector control, and the protein expression level of synaptophysin was measured by Western blot analysis. The results demonstrate that overexpression of Siah-1A leads to a significant decrease in the steady-state level of synaptophysin expression (Fig. 7A). The decrease in synaptophysin level is not due to a difference in the transfection efficiency, because similar results were obtained when the transfection efficiency was normalized using a cotransfected luciferase reporter gene (data not shown).
To further determine whether Siah proteins regulate the degradation of synaptophysin, we examined the effect of overexpression of Siah-1A on synaptophysin protein turnover by pulse-chase experiments (Fig. 6). In cells co-transfected with synaptophysin and control vector, the half-life of synaptophysin was ϳ6 h (Fig. 6), which is consistent with the half-life of endogenous synaptophysin in cultured hippocampal neurons (62). In contrast, when synaptophysin was co-expressed with Siah-1A, the half-life of synaptophysin protein was reduced to 1.8 h (Fig. 6). Taken together, these results indicate that Siah proteins have the ability to regulate synaptophysin turnover.
Siah Regulates Synaptophysin Degradation via the Ubiquitin-proteasome Pathway-Next, we sought to determine whether the enhanced degradation of synaptophysin by Siah proteins is mediated by the proteasome pathway. HeLa cells that have been co-transfected with synaptophysin and Siah-1A were treated for 8 h with various inhibitors of proteolytic pathways, and the synaptophysin levels were then analyzed by Western blotting. As shown in Fig. 7A, the enhanced degradation of synaptophysin by Siah-1A overexpression is blocked by MG132, a potent inhibitor of proteasome function (63). A similar effect was also observed when cells were treated with lactacystin, an irreversible inhibitor of the proteasome pathway (data not shown) (63). In contrast, E64, an inhibitor of lysosomal cysteine proteases, had no effect.
Because proteasome-dependent proteolysis involves the ubiquitination of target proteins, we investigated whether Siah-1A accelerates the degradation of synaptophysin by promoting the ubiquitination of synaptophysin. Myc-tagged synaptophysin was co-expressed in CHO cells along with HAtagged ubiquitin in the absence or presence of exogenous Siah-1A or an N-terminally truncated Siah-1A (Siah-1A ⌬N, amino acids 137-282). Cell lysates were subjected to immunoprecipitation with an anti-myc antibody followed by immunoblotting with an anti-HA antibody to detect ubiquitin-conjugated synaptophysin (Fig. 7B). In the absence of exogenous Siah proteins, synaptophysin was ubiquitinated, because the immunoprecipitate contained high molecular weight smears detected by the anti-HA antibody immunoblotting. Overexpression of Siah-1A enhanced the ubiquitination of synaptophysin, because increased levels of HA-tagged ubiquitin were detected on synaptophysin. Conversely, overexpression of Siah-1A ⌬N, a dominant negative form of Siah-1A that lacks the RING fingercontaining N-terminal region, blocked the ubiquitination of synaptophysin. Taken together, these data indicate that Siah proteins target synaptophysin for ubiquitin-mediated degradation via the 26 S proteasome.
Siah Associates with the E2 Ubiquitin-conjugating Enzyme UbcH8 and Regulates the Degradation of Endogenous Synaptophysin in PC12 Cells-To address the question whether Siah proteins could regulate the degradation of endogenous synaptophysin, we examined the effect of overexpression of myc-Siah-2 on the protein level of endogenous synaptophysin in PC12 cells. Cell lysates were prepared from PC12 cells transfected with pcDNA3-myc-Siah-2 (35) or a pcDNA3-myc vector control, and the steady-state levels of myc-Siah-2 and synaptophysin in these lysates were determined by Western blot analysis (Fig. 8A). Consistent with previous reports (35,37), myc-tagged Siah-2 was found to be expressed at a relatively low level in transfected PC12 cells, perhaps as a result of selfregulating its own stability (see "Discussion"). Comparison with the control transfection reveals that the expression of exogenous Siah-2 significantly down-regulated the protein level of endogenous synaptophysin. To further determine whether the down-regulation effect of Siah-2 overexpression is mediated via the ubiquitin-proteasome degradation pathway, PC12 cells expressing myc-Siah-2 were treated with various inhibitors of protein degradation. Comparison of the synaptophysin levels in treated versus untreated cells shows that the down-regulation effect of Siah-2 overexpression is blocked by the proteasome inhibitor MG132 but not by the lysosomal protease inhibitor E64 (Fig. 8A). These results suggest that Siah proteins regulate the degradation of endogenous synaptophysin in PC12 cells via the ubiquitin-proteasome pathway. As previously reported for Siah-1 (35,37), the expression levels of myc-Siah-2 were increased upon inhibition of the proteasome activity by MG132 (Fig. 8A), suggesting that the stability of Siah-2 itself is controlled by the proteasome pathway. Interestingly, Siah has recently been shown to promote its own ubiquitination (64), and this autoubiquitination of Siah could lead to the self-regulation of its own degradation.
A likely mechanism by which Siah regulates the ubiquitination and degradation of synaptophysin is that Siah may serve as an E3 ubiquitin-protein ligase for synaptophysin. E3 ligases determine the specificity of protein degradation by recognizing a unique set of substrates and cooperating with a specific E2 ubiquitin-conjugating enzyme to catalyze the ubiquitination (65). Although Sina has been shown to interact with the E2 enzyme UbcD1 in Drosophila (30), the one or more cognate E2 ubiquitin-conjugating enzymes for mammalian Siah proteins remain to be clarified. To test whether Siah interacts with a specific E2 enzyme, we expressed various HA-tagged E2 enzymes (66) by transient transfection into HeLa cells along with myc-tagged Siah-2. The interaction of these E2 enzymes with Siah-2 was examined by immunoprecipitation with an antimyc antibody followed by immunoblotting with an anti-HA antibody (Fig. 8B). The results reveal that Siah-2 specifically interacts with UbcH8, an E2 enzyme that is abundantly expressed in the central nervous system (67) but not with other E2 enzymes examined, namely UbcH5 and UbcH7. These data suggest that Siah may function as E3 ubiquitin ligase to regulate the ubiquitination and degradation of synaptophysin in conjunction with the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8. DISCUSSION Although synaptophysin has been implicated in neurotransmitter release, and decreased synaptophysin levels have been associated with several neurodegenerative diseases, the molecular mechanism underlying the degradation and turnover of synaptophysin remains unsolved. In the current study, we demonstrate that Siah proteins interact with synaptophysin and regulate the degradation of synaptophysin via the ubiquitin-proteasome pathway. It has been previously shown that Sina/Siah proteins promote ubiquitin-proteasome-dependent degradation of several nuclear proteins (29,30,68) as well as DCC, a neuronal plasma membrane protein (35). The evidence presented here indicates that Siah proteins also regulate the degradation of synaptophysin, a synaptic vesicle membrane protein.
Sina/Siah proteins are widely expressed in the embryo and adult tissues, and a high level of Siah-2 expression is found in certain neuronal tissues, including olfactory epithelium, retina, and forebrain (25,31,33,69,70). Our subcellular fractionation studies have shown that Siah proteins exist in both cytosolic and membrane-associated pools. Furthermore, immunofluorescence studies reveal that, in NGF-differentiated PC12 cells, Siah proteins are primarily localized in the cell body and neuritic processes, where they are associated with synaptic-like microvesicles and early endosomes. This localization pattern suggests that Siah proteins may play an important role in regulation of the expression levels of synaptophysin and perhaps other synaptic vesicle proteins at nerve terminals.
The ubiquitin-proteasome pathway consists of two major steps: The conjugation of ubiquitin to the substrate and subsequent degradation of the ubiquitinated protein by the 26 S proteasome (71). Ubiquitin conjugation involves sequential reactions in which ubiquitin is first activated by a ubiquitinactivating enzyme (E1), then transferred to a ubiquitin-conjugating enzyme (Ubc or E2), and finally ligated to the substrate by a ubiquitin-protein ligase (E3) (65,71,72). The specificity of ubiquitin conjugation is conferred by the E3, which binds the substrate protein and cooperates with the E2 enzyme to catalyze the covalent attachment of ubiquitin to the substrate. Our data support a model in which Siah regulates the degradation of synaptophysin by acting as an E3 ubiquitin-protein ligase to catalyze the ubiquitination of synaptophysin in conjunction with the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8. Formation of a polyubiquitin chain then targets synaptophysin for degradation by the 26 S proteasome.
The structural requirement underlying the recognition of substrates by E3 ligases is largely unknown. An E3 can recognize several substrates (73), which is clearly the case with Siah FIG. 8. Siah binds selectively to UbcH8 and down-regulates endogenous synaptophysin in PC12 cells. A, Siah-2 promotes synaptophysin degradation via the ubiquitin-proteasome pathway. PC12 cells were transfected with pcDNA3-myc-Siah-2 or the pcDNA3-myc vector. Twenty-four hours later, cells were incubated for 8 h at 37°C with Me 2 SO, proteasome inhibitor MG132 (20 M in Me 2 SO), or cysteine protease inhibitor E64 (50 M). Cells were then lysed, and equal amounts of protein from each lysate were analyzed by immunoblotting for synaptophysin, myc-Siah-2, and actin. B, Siah-2 interacts specifically with UbcH8. HeLa cells were co-transfected with pcDNA3-myc-Siah-2 and pRK5-HA-UbcH5, pRK5-HA-UbcH7, or pRK5-HA-UbcH8. Cell lysates of transfected cells were subjected to immunoprecipitation with an anti-myc antibody. The immunoprecipitates were then analyzed by immunoblotting with antibodies against HA, myc, or actin. Syp, synaptophysin.
proteins. Previous studies have shown that, although the Nterminal RING finger domain of Siah proteins is required for interacting with E2s, the C-terminal region is involved in binding substrates such as DCC and N-CoR, although the exact binding site has not been described (37). Our deletion results indicate that the synaptophysin-binding site resides within the region between residues 225 and 278 of Siah-2. This region (225-278) of Siah-2 is included in the previously reported substrate-binding regions (108 -324 for N-CoR; 184 -324 for DCC) (35,68). It will be interesting to determine whether the binding site for these substrates also falls between residues 225 and 278 of Siah-2.
Synaptophysin has a unique C-terminal cytoplasmic tail that contains nine copies of an imperfect pentapeptide repeat, YG-P(Q)QG. The function of these repeats is not understood, although they have been proposed to mediate protein-protein interactions (1,3). By using deletion analysis, we have shown that the C terminus of synaptophysin containing the last three repeats (residue 278 -307) is responsible for binding Siah-1A. The amino acid sequence (278 -307) of synaptophysin C terminus is well conserved evolutionarily, further supporting the functional significance of this region. Moreover, this region is also well conserved in synaptoporin, a 37-kDa protein with 58% amino acid identity to synaptophysin (74,75), suggesting that synaptoporin could also be a substrate of Siah proteins. In support of this hypothesis, our preliminary studies have shown that synaptoporin physically interacts with Siah proteins. 2 The ubiquitin-proteasome pathway plays a crucial role in the degradation of proteins involved in a variety of cellular processes, including differentiation, proliferation, and apoptosis (65,71,76). However, the role of the ubiquitin-proteasome pathway in the degradation of synaptic vesicle-associated proteins remains poorly characterized, despite the identification of ubiquitin in synaptic terminals (77,78). In this study, we have shown that synaptophysin, an integral membrane protein of synaptic vesicles, is ubiquitinated and degraded by the proteasome pathway. Furthermore, the ubiquitin-dependent degradation of synaptophysin is regulated by Siah proteins, a family of putative E3 ligases. Interestingly, Parkin, a protein implicated in the pathogenesis of familial Parkinson's disease, has been shown to act as an E3 ligase for the degradation of CDCrel-1, a member of the septin family that is associated with synaptic vesicles (66). Moreover, Parkin has recently been reported to be an E3 ligase for the degradation of ␣-synuclein, a synaptic vesicle-associated protein whose mutations have been linked to familial Parkinson's disease (79).
It is becoming increasingly clear that malfunctions of the ubiquitin-proteasome pathway are critically involved in the pathogenesis of a number of neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, and Huntington's disease (65,80). Since altered synaptophysin expression levels have been linked to several neurological disorders, dysregulation of synaptophysin degradation by Siah proteins may represent a mechanism contributing to the pathophysiology of these diseases. Interestingly, Siah proteins have been reported to be up-regulated during apoptosis (34,69,81). Given that overexpression of Siah proteins promotes the degradation of synaptophysin as well as DCC, it is possible that increased Siah expression promotes the degeneration of synaptic terminals and the death of neurons via accelerated degradation of these and other yet to be identified substrate proteins. Further studies of Siah proteins and their protein-protein interactions are likely to yield novel insights into the mechanisms by which the ubiquitin-proteasome degradation pathway regulates the development, function, and degeneration of nerve terminals.