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J. Biol. Chem., Vol. 276, Issue 44, 41112-41119, November 2, 2001

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Formation of Crystalloid Endoplasmic Reticulum Induced by Expression of Synaptotagmin Lacking the Conserved WHXL Motif in the C Terminus

STRUCTURAL IMPORTANCE OF THE WHXL MOTIF IN THE C2B DOMAIN^*

Mitsunori Fukuda^§, Akitsugu Yamamoto^¶, and Katsuhiko Mikoshiba

From the  Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, the ^¶ Department of Physiology, Kansai Medical University, Moriguchi, Osaka 570-8506, and the  Department of Basic Medical Science, Division of Molecular Neurobiology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

Received for publication, July 3, 2001, and in revised form, August 23, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Synaptotagmin (Syt) is a family of type I membrane proteins that consists of a single transmembrane domain, a spacer domain, two Ca²⁺-binding C2 domains, and a short C terminus. We recently showed that deletion of the short C terminus (17 amino acids) of Syt IV prevented the Golgi localization of Syt IV proteins in PC12 cells and induced granular structures of various sizes in the cell body by an unknown mechanism (Fukuda, M., Ibata, K., and Mikoshiba, K. (2001) J. Neurochem. 77, 730-740). In this study we showed by electron microscopy that these structures are crystalloid endoplasmic reticulum (ER), analyzed the mechanism of its induction, and demonstrated that: (a) mutation or deletion of the evolutionarily conserved WHXL motif in the C terminus of the synaptotagmin family (Syt C) destabilizes the C2B domain structure (i.e. causes misfolding of the protein), probably by disrupting the formation of stable anti-parallel -sheets between the -1 and -8 strands of the C2B domain; (b) the resulting malfolded proteins accumulate in the ER rather than being transported to other membrane structures (e.g. the Golgi apparatus), with the malfolded proteins also inducing the expression of BiP (immunoglobulin binding protein), one of the ER stress proteins; and (c) the ERs in which the Syt C proteins have accumulated associate with each other as a result of oligomerization capacity of the synaptotagmin family, because the Syt IC mutant, which lacks oligomerization activity, cannot induce crystalloid ER. Our findings indicate that the conserved WHXL motif is important not only for protein interaction site but for proper folding of the C2B domain.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Synaptotagmin I (Syt I)¹ is an integral membrane protein of synaptic vesicles and a key molecule that regulates various steps of the synaptic vesicle cycle. It consists of a short intravesicular N terminus, a single transmembrane domain, a spacer domain, a C2A domain, a C2B domain, and a short C terminus (reviewed in Refs. 1-4). Each domain of Syt I has been shown to be crucial to expression of its functions and to have a distinct role in the synaptic vesicle cycle. For instance, the short C terminus (WHXL motif) of Syt I is involved in synaptic vesicle docking to the presynaptic plasma membrane, probably by binding to certain plasma membrane receptors, such as neurexins (5). The Ca²⁺ binding activity of the C2A domain is crucial for synaptic vesicle fusion to the presynaptic plasma membrane, because antibodies to the C2A domain that inhibits Ca²⁺/phospholipid or Ca²⁺/syntaxin binding to it interfere with the fusion step of secretory vesicles (6-11). The Syt I C2A domain is also important for neurite outgrowth and the axonal repair process (12-15). The C2B domain is involved in recycling of synaptic vesicles, probably by binding to the clathrin assembly protein AP-2 (16-18). The Ca²⁺-dependent self-oligomerization activity of the C2B domain has also been suggested to be involved in the fusion step of synaptic vesicles (19-22), because a Tyr-312 to Asn substitution in the C2B domain of Syt II, corresponding to the Drosophila AD3 mutation, impairs Ca²⁺-dependent self-oligomerization activity (20, 21), and inositol 1,3,4,5-tetrakisphosphate, a strong blocker of neurotransmitter release, inhibits Ca²⁺-dependent self-oligomerization activity of the C2B domain in vitro (20, 23-25). In addition, the phenotypes of Syt I mutants of Drosophila, Caenorhabditis elegans, and mice strongly support the above multiple functions of Syt I in the synaptic vesicle cycle (26-32).

Synaptotagmins are now known to form a large family of integral membrane proteins in both vertebrates and invertebrates (1, 33, 34), and it has been suggested that they regulate vesicular trafficking, because Syts II-XIII basically share the same domain structure with Syt I (i.e. a transmembrane domain, spacer domain, two C2 domains, and short C terminus). One of these domains, the N-terminal region of the C terminus, is highly conserved across phyla as well as different isoforms (Syts I-XIII), and it has been suggested to have functions common to the synaptotagmin family (5, 35). In our previous study, we showed that the WHXL motif in the C terminus of Syts I-XI is essential for plasma membrane association in PC12 cells and that the WHXL motif of Syt I regulates synaptic vesicle docking to the presynaptic plasma membrane in the squid giant synapse (5). In this study we report a novel role of the WHXL motif of the synaptotagmin family in proper folding of the molecules. We have shown by deletion and mutation analyses that the WHXL motif is essential for correct folding of the C2B domain (i.e. the WHXL motif is located in the -8 strand of the C2B domain rather than being an independent domain; see Fig. 3A). Expression of synaptotagmin mutants lacking the WHXL motif in cultured cells induced crystalloid endoplasmic reticulum (ER) structures (Refs. 36 and 37, and references therein) due to the abnormal folding of the proteins. Based on these results, we discuss the structural importance of the -8 strand of the C2 domains of synaptotagmins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Recombinant Taq DNA polymerase and restriction enzymes were obtained from Toyobo Biochemicals (Tokyo, Japan). Mouse monoclonal antibody (M2) and rabbit polyclonal antibody against FLAG peptide were obtained from Sigma Chemical Co. (St. Louis, MO) and Zymed Laboratories Inc. (San Francisco, CA), respectively. Horseradish peroxidase (HRP)-conjugated anti-T7 tag antibody and anti-T7 tag antibody-conjugated agarose were from Novagen (Madison, WI). HRP-conjugated anti-FLAG tag antibody was from Sigma. Anti-TGN38 antibody was from Transduction Laboratories (Lexington, KY). Anti-BiP (immunoglobulin binding protein, also called Grp78, a 78-kDa glucose-regulated protein) antibody was from StressGen Biotechnologies Corp. (Victoria, BC, Canada). All other chemicals were commercial products of reagent grade. Solutions were made up in deionized water prepared with an Elix10 water purification system and Milli-Q Biocel A10 system (Millipore Corp., Bedford, MA).

Plasmid Construction-- pEF-FLAG(or T7)-Syts I-XI, -Syts I-XIC, -T7-N-Gly-Syt VIII, -T7-N-Gly-Syt IX, -T7-N-Gly-Syt VIIIC, -T7-N-Gly-Syt IXC, and -FLAG-Syt IVC2B were essentially produced as described previously (5, 38-41). pEF-FLAG-Syt IV414-425, -Syt IV419-425, -Syt IV423-425, -Syt IV423-425(AAA), -Syt IV329-425, -Syt IV361-425, and -Syt IV(C2A)C were constructed by the polymerase chain reaction using the following sets of primers with appropriate restriction enzyme sites (underlined) and/ or termination codons (boldface letters): 5'-GCACTAGTTCACTTAGCAATTTGTC(AT)CC-3' (414/419 primer; antisense; amino acid residues 413-418), 5'-GCACTAGTTCAGAGCATGTGCCACT-3' (423 primer; antisense; amino acid residues 418-422), 5'-GCACTAGTTCAGGCCATGGCCGCCTTAGCAATTTG-3' (423(AAA) primer; antisense; amino acid residues, 415-422), 5'-GCACTAGTCATTTGACGTAGGGATCTGAA-3' (329 primer, antisense; amino acid residues 322-328), 5'-GCACTAGTCAAAAGACAAACAGTTCATTG-3' (361 primer, antisense; amino acid residues 354-360), and 5'-GCACTAGTCACATTAACATTTTTCCATC-3' (C2AC primer; antisense, amino acid residues 268-273).

pEF-FLAG-Syt IVC(341-347) and -Syt IVC(K341A, K342A,K346A) (KA) were essentially produced by means of two-step polymerase chain reaction techniques as described previously (25), using the following pairs of oligonucleotides: C2A primer, 5'-CGGATCCCAAGAGAAGCTGGGGACACT-3' and K/A primer, 5'-GCGTGCACTTCGCAACGTGAGTCGCCGCTTTAGAG-3' or K primer, 5'-GCGTGCACTTAGAGATTCTCTTCTT-3' (left half), ApaLI primer, 5'-GCGTGCACTCCCAACGCAGTGTTC-3' and C primer, 5'-GCACTAGTTCAGATCTCCTTCCAGTGCCC-3' (right half). pEF-FLAG-Syt IV(C2A-1A3) and -Syt IV(C2A-1) were similarly constructed using the following pairs of mutagenic oligonucleotides: C2A-1A3-5' primer, 5'-GACCAGAGCTGCAGCCCGGCCAGAAGACT-3' and C2A-1A3-3' primer, 5'-TCTGGCCGGGCTGCAGCTCTGGTCTCTCT-3'; and C2A-1-5' primer, 5'-ACCGCGGCCAGAAGACTTCTT-3' and C2A-1-3' primer, 5'- CCGCGGTACAAACACGCTCACTGTGGTG-3'. The resulting Syt IV mutant fragments were subcloned into the NotI site of the modified pEF-BOS mammalian expression vector (24, 42). All constructs were verified by DNA sequencing using a Hitachi SQ-5500 DNA sequencer. Plasmid DNA was prepared using Wizard-mini preps (Promega, Madison, WI) or Qiagen (Chatsworth, CA) Maxi prep kits.

Immunocytochemistry-- Glass-bottom dishes (35 mm-dish, MatTek Corp.) were coated with collagen type IV (Becton Dickinson, Bedford, MA). PC12 cells (0.8-1 × 10⁵ cells, the day before transfection) were cultured on these dishes in Dulbecco's modified Eagle's medium containing 10% horse serum and 10% fetal bovine serum at 37 °C under 5% CO₂. Transfection was achieved by using the LipofectAMINE Plus reagent according to the manufacturer's instructions (Life Technologies Inc., Gaithersburg, MD). Three days after transfection, PC12 cells were fixed, permeabilized, and stained with both rabbit anti-FLAG antibody (1/1000 dilution) and anti-TGN38 mouse monoclonal antibody (1/500 dilution) or anti-BiP mouse monoclonal antibody (1/200 dilution), as described previously (43, 44). Immunoreactivity was analyzed with a fluorescence microscope (TE300, Nikon, Tokyo, Japan) attached to a laser confocal scanner unit CSU 10 (Yokogawa Electric Corp., Tokyo, Japan) and HiSCA charge-coupled device camera (C6790, Hamamatsu Photonics, Hamamatsu, Japan). Images were pseudo-colored and superimposed with Adobe Photoshop software (Version 4.0).

Electron Microscopy-- PC12 cells cultured on collagen-coated plastic coverslips (Sumitomo Bakelite, Tokyo, Japan) were fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 2 h. After washing in the buffer, cells were post-fixed in 1% OsO₄ in 0.1 M sodium phosphate buffer (pH 7.4) for 1 h. The cells were then washed in distilled water, incubated with 50% ethanol for 10 min, and stained with 2% uranyl acetate in 70% ethanol for 2 h. The cells were further dehydrated with a graded series of ethanol and embedded in epoxy resin. Ultra-thin sections were doubly stained with uranyl acetate and lead citrate and then examined under a Hitachi H7000 electron microscope.

Synaptotagmin Oligomerization Assay-- pEF-T7-Syts and pEF-FLAG-Syts were cotransfected into COS-7 cells (5 × 10⁵ cells the day before transfection/10-cm dish) as described previously (45). Three days after transfection, cells were harvested and homogenized in 50 mM HEPES-KOH, pH 7.2, 250 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 10 µM pepstatin A. The proteins expressed were solubilized with 1% Triton X-100, immunoprecipitated with anti-T7 tag antibody-conjugated agarose, and analyzed by 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) as previously described (38, 40, 45). Co-immunoprecipitated FLAG-Syts were first detected with HRP-conjugated anti-FLAG antibody (1/10,000 dilution). The same blots were stripped and reprobed with HRP-conjugated anti-T7 tag antibody to ensure loading of the same amounts of T7-Syt proteins (1/10,000 dilution).

Endoglycosidase H Digestion of T7-N-Gly-synaptotagmins-- PC12 cells (4 × 10⁶ cells, the day before transfection) were cultured on 10-cm collagen type I-coated dishes (Becton Dickinson) in Dulbecco's modified Eagle's medium containing 10% horse serum and 10% fetal bovine serum at 37 °C under 5% CO₂. Transfection was achieved by using the LipofectAMINE Plus reagent as described above. Immunoprecipitation of T7-Syts by anti-T7 tag antibody-conjugated agarose was performed as described previously (38, 39, 45). The beads were resuspended in 30 µl of the buffer (50 mM sodium acetate, pH 5.3, 0.1% SDS, 0.5% IGEPAL CA-630 (Sigma Chemical Co.), 0.1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 10 µM pepstatin A) and then divided into two microtubes. After denaturation by boiling for 3 min and cooling to 37 °C, 5 milliunits of endoglycosidase H (Roche Diagnostics, Mannheim, Germany) was added to one tube, and the mixtures were incubated for 1 h at 37 °C (41). Reactions were stopped by adding SDS sample buffer and boiling for 3 min. Proteins were subjected to 10% SDS-PAGE and immunoblotting with HRP-conjugated anti-T7 tag antibody as described previously (38-40).

Trypsin Digestion of Recombinant C2B Domain of Synaptotagmin IV-- Syt IV-C2B423-425 and mutant Syt IV-C2B423-425(AAA) fused to glutathione S-transferase (GST) were prepared by a standard method (24, 46), and after incubating 4 µg of GST fusion proteins in 10 mM Tris-HCl, pH 7.5, and 100 mM NaCl with 1 ng/µl trypsin (Sigma Chemical Co.) for 0-30 min at 25 °C, the reactions were stopped by addition of SDS sample buffer and boiling for 3 min. Samples were then analyzed by 12.5% SDS-PAGE and staining with Coomassie Brilliant Blue R-250.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Induction of Crystalloid ER by Expression of Synaptotagmin IV Lacking the WHXL Motif-- In the course of determining the Golgi localization signal of Syt IV in PC12 cells, we found that the deletion of the short C terminus (amino acid residues 409-425) prevented the Golgi localization of Syt IV and induced large granular structures of various sizes (Ref. 41 and Fig. 1, A and C). Interestingly, these granular structures in which Syt IVC proteins had accumulated were easily identified on light-field micrographs (arrowheads in Fig. 1D). Because they were also positive with anti-BiP antibody, a marker for the ER, we suspected that they represented crystalloid ER (36, 37; see also Fig. 1, E-G, arrows). Electron microscopic observation of the cells expressing Syt IVC revealed a unique membranous structure characteristic to "crystalloid ER" (36, 37), which is composed of tightly packed smooth ER cisternae (Fig. 3H, arrowheads) or tubules (Fig. 3H, arrows). The crystalloid ER was occasionally continuous to rough ER cisternae (Fig. 3H, small arrows).

View larger version (62K): [in this window] [in a new window]   Fig. 1.   Deletion of the short C terminus prevents Golgi localization of synaptotagmin IV and induces crystalloid ER. FLAG-Syt IV (A, B) and -Syt IVC (C-H) proteins were expressed in PC12 cells as described under "Experimental Procedures." PC12 cells were fixed, permeabilized, and stained with anti-FLAG rabbit antibody (green in A, C, E, and G) and anti-BiP antibody (red in F and G). B and D are light-field micrographs of A and C, respectively. The inset in D is a superposition of C and D. Note that the granules could be detected by light-field microscopy (arrowheads in D) and that they colocalized with BiP (arrows in E-G). H, electron micrograph of PC12 cells expressing FLAG-Syt IVC proteins. The crystalloid ER is composed of tightly packed smooth ER cisternae (arrowheads in H) or tubule (arrows in H). The crystalloid ER was occasionally continuous to rough ER cisternae (small arrows in H). DCV, dense-core vesicle; RER, rough ER; M, mitochondria. Scale bars in G and H indicate 20 µm and 500 nm, respectively.

To identify the amino acid residues responsible for the induction of crystalloid ER by expression of Syt IVC, we constructed various additional C-terminal deletion mutants (Fig. 2A). After deletion of more than seven amino acids from the C terminus (FLAG-Syt IV419-425), we observed mis-sorting of Syt IV mutant proteins and formation of crystalloid ER of various sizes within the cell body (Fig. 2C, 414-425, 419-425 in green and TGN38 in red), but deletion of three amino acids did not affect the Golgi localization of Syt IV (Fig. 2C, 423-425, arrowheads). When the conserved WHML (amino acids 419-422) sequences were mutated to AAMA, expression of FLAG-Syt IV423-425(AAA) induced granule formation similar to that of Syt IVC (Fig. 2C). Thus, the WHXL sequences are in some way related to the formation of crystalloid ER and the Golgi localization of Syt IV. Because deletion of the whole C2B domain (FLAG-Syt IVC2B) attenuated the crystalloid ER formation induced by deletion of the C terminus (C) and Syt IVC2B proteins were localized in the Golgi (Fig. 2C, arrowhead in center panel), the C2B domain was further systematically deleted from the C terminus, and the putative effector domain of the C2B domain was deleted or mutated to clarify the relationship between the C2B domain and the WHXL sequences (Fig. 2C) (25, 47, 48). These mutations and deletions, however, failed to restore the Golgi localization of Syt IVC proteins (Fig. 2C, 329-425, 361-425, C(341-347), and C(KA) in green and TGN38 in red).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Expression of synaptotagmin IV lacking the WHXL motif induces crystalloid ER in PC12 cells. A, schematic representation of the C-terminal mutants of Syt IV. B, schematic representation of the C2B deletion mutants of Syt IV. FLAG tags, the transmembrane domain (TM), and two C2 domains are represented by black boxes, open boxes, and hatched boxes, respectively. The double dots and dashes indicate amino acids identical to the top sequence and deletions, respectively. Amino acid substitutions are indicated by the single-letter amino acid code below the wild-type sequences (bottom lines in A and B). Amino acid numbers, except for the FLAG tag sequence, are given on the right. C, subcellular localization of the C-terminal and C2B deletion mutants of FLAG-Syt IV414-425, -Syt IV419-425, -Syt IV423-425, -Syt IV423-425(AAA), -Syt IVC2B, -Syt IV329-425, -Syt IV361-425, -Syt IVC(341-347), and -Syt IVC(KA) were expressed in PC12 cells as described under "Experimental Procedures." PC12 cells were fixed, permeabilized, and stained with both anti-FLAG rabbit (green) and anti-TGN38 antibodies, a marker for the TGN (red). Note that only FLAG-Syt IV423-425 and -Syt IVC2B overlapped well with TGN38 (arrowheads). Scale bar indicates 20 µm.

The Formation of Crystalloid ER Structures by Expression of Other Synaptotagmins Lacking the WHXL Motif-- The WHXL motif is highly conserved among the C-terminal type tandem C2 proteins, including the synaptotagmin family, rabphilin-3A, the Doc2 family, and the Slp family (synaptotagmin-like protein) (35, 49, 50) and was proposed to be an independent domain (4), because the short C terminus of these proteins interacts with polymorphic plasma membrane receptors, neurexins, in vitro (33, 49, 51-54). However, recent demonstration of the three-dimensional structure of the C2B domain of Syt III (55) and rabphilin-3A (56) has provided strong evidence against this notion, and the WHXL motif must be a part of the C2B domain rather than an independent domain. The C2B domain of Syt III is composed of an eight-stranded anti-parallel -sandwich structure consisting of four-stranded -sheets (anti-parallel pairing of -1 and -8, -2 and -5, -3 and -4, and -6 and -7 strands), the same as the Syt I C2A domain (reviewed in Ref. 57; see also Fig. 3A). The major difference between the C2A and C2B domains is that the C2B domain contains an additional -helix region between the -7 and -8 strands (Fig. 3A). It should be noted that this -helix region was previously thought to be a -8 strand of the C2B domain (Fig. 3B, broken arrow) (55, 56). Based on these findings, together with the fact that crystalloid ER is induced by expression of malfolded proteins (58), we hypothesized that deletion of a portion of the -8 strand (the WHXL motif) of the C2B domain destabilizes the C2B domain structure (i.e. the C2B domain lacking the WHXL motif cannot fold correctly) and induces the crystalloid ER structures.

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Proposed structure of the C2B domain of synaptotagmins. A, schematic representation of the C2B domain of synaptotagmin IV (modified from Ref. 57). The arrows represent eight strands of the C2B domain (1-8). Three loops are formed at the tip of the -sandwich structure, and two of them are involved in Ca2+-binding in the C2A domain of Syt I (57). The overall structure of the C2B domain is quite similar to the C2A domain, but an additional -helix is present between the -7 and -8 strands in the C2B domain (55, 56). Note that the N-terminal -1 strand and C-terminal -8 strand form a pair to create an anti-parallel -sheet. B, sequence alignment of the -1 and -8 strands (solid arrows) and the -helix (broken line) of the Syts I-XIII C2B domains (33, 38, 55). Asterisks indicate the conserved WHXL motif (5). The number signs indicate the positions of amino acid substitutions (GEL-to-AAA) in the Syt IV(C2B-1A3) mutant (see also Fig. 5A). Note that the -helix region was previously thought to be a -8 strand of the C2B domain by analogy with the C2A domain (8', broken arrow at the bottom) (57).

If this hypothesis were true, deletion of the -8 strand of the C2B domain of other synaptotagmin isoforms should induce the same crystalloid ER structures, because the WHXL motif is highly conserved in the synaptotagmin family (Fig. 3B). As expected, expression of Syts I-XIC proteins also induced crystalloid ER-like structures in PC12 cells (Fig. 4A, arrowheads and data not shown), although the appearance of the crystalloid ER-like structures differs according to the Syt isoform (5-50% of total cells). Interestingly, expression of an Syt IC mutant that lacks oligomerization activity (Fig. 4B; Syt Ispacer/C) (59) failed to induce crystalloid ER-like structures, despite the absence of the WHXL motif (Fig. 4A, right upper panel).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Induction of crystalloid ER-like structures by C-terminal truncation of the synaptotagmin family. A, FLAG-Syt IC, -Syt Ispacer/C, -Syt VIC, and -Syt XIC were expressed in PC12 cells as described under "Experimental Procedures." PC12 cells were fixed, permeabilized, and stained with anti-FLAG antibody. Note that the crystalloid ER-like structures were induced by expression of Syt IC, Syt VIC, and Syt XIC (arrowheads), but not Syt Ispacer/C, which did not exhibit Ca2+-independent oligomerization activity (59) (see also B). Scale bar indicates 20 µm. B, oligomerization activity of Syt IC (or Syt Ispacer/C). pEF-T7-Syt I and FLAG-Syt IC (or -Syt Ispacer/C) were co-transfected into COS-7 cells. Association between T7-Syt I and FLAG-Syt IC (or -Syt Ispacer/C) was evaluated by immunoprecipitation with anti-T7 tag antibody-conjugated agarose (IP) as described previously (38, 45). Top panel, total expressed FLAG-Syt IC and -Syt Ispacer/C (1/40 volumes of reaction mixtures; input) used for immunoprecipitation. Middle panel, co-immunoprecipitated FLAG-tagged proteins (blot: anti-FLAG). Lower panel, immunoprecipitated T7-Syt I (blot: anti-T7). The arrow indicates the SDS-resistant dimer of Syt IC. The positions of the molecular weight (Mr) markers (×103) are shown on the left.

Next, we focused on the -1 strand of the C2B domain of Syt IV, because the N-terminal -1 strand and C-terminal -8 strand pair to form anti-parallel -sheets (Fig. 3A). Deletion of the -1 strand of the C2B domain of Syt IV (C2B-1, Fig. 5) or substitution of AAA for the conserved G(E/D)(L/I) sequence of the -1 strand of Syt IV (C2B-1A3) induced crystalloid ER-like structures, the same as Syt IVC, suggesting that pairing of the -1 and -8 strands is important for proper folding of the C2B domain. These results, together with the fact that the basic structure of both the C2A and C2B domains is the same (55-57), prompted to us to examine whether deletion of the -8 strand of the C2A domain would also induce crystalloid ER. As expected, expression of Syt IV(C2A)C proteins induced the same crystalloid ER-like structures (Fig. 5B, top panel).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Expression of synaptotagmin IV lacking the -1 strand of the C2B domain or -8 strand of the C2A domain induces crystalloid ER-like structures in PC12 cells. A, schematic representation of the -1 (or -8) strand-mutants of Syt IV. B, subcellular localization of the -1 and -8 strand-mutants of Syt IV. FLAG-Syt IV(C2A)C, -Syt IV(C2B-1), and -Syt IV(C2B-1A3) were expressed in PC12 cells as described under "Experimental Procedures." PC12 cells were fixed, permeabilized, and stained with anti-FLAG antibody. In all cases, crystalloid ER-like structures were induced. Scale bar indicates 20 µm.

Incomplete Post-translational Modifications of Syt C Proteins in PC12 Cells-- Vertebrate Syts I and II contain a single N-glycosylation site in the N-terminal intravesicular domain (24, 39, 41, 51, 60), and the N-liked sugars are further converted to complex oligosaccharides in the Golgi of PC12 cells (41). Because the dense-core vesicles where Syt I is abundant are thought to be derived from the TGN (trans-Golgi network) in PC12 cells, we decided to investigate whether Syt IC proteins pass through the Golgi and TGN or are only retained in the ER. To do so, we used endoglycosidase H, which cleaves unmodified N-linked sugars but not complex oligosaccharides (41). The N-linked sugars of the wild-type T7-Syt I, -Syt II, -N-Gly-Syt VIII, and -N-Gly-Syt IX in PC12 cells were further modified and were resistant to endoglycosidase H treatment, whereas the mutant proteins (Syt C) were mostly sensitive to endoglycosidase H (Fig. 6, A and B). By contrast, the N-linked sugars of both the wild-type and mutant proteins were cleaved by N-glycosidase F, which cleaves all N-linked sugars, regardless of modifications (data not shown). These results indicate that the newly synthesized Syt C proteins had been correctly inserted into the ER membrane and had been retained in the ER without being transported to the Golgi, where complex modification occurs.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Analysis of T7-Syts IC, IIC, T7-N-Gly-Syts VIIIC, and IXC by endoglycosidase H treatment. A, treatment of T7-Syt I, -Syt IC, -Syt II, and -Syt IIC with endoglycosidase H (endo H). T7-Syts were expressed in PC12 cells and immunoprecipitated by anti-T7 tag antibody-conjugated agarose as described previously (38, 45). Immunoprecipitants were exposed to or not exposed to glycosidase, subjected to 10% SDS-PAGE, and then analyzed by immunoblotting with HRP-conjugated anti-T7 tag antibody. Note that Syts I and II were resistant to endoglycosidase H (broken boxed), but Syts IC and IIC were highly sensitive to endoglycosidase H. The results shown are representative of three independent experiments. The positions of the Mr markers (×103) are shown on the left. B, proportion of the endoglycosidase H-resistant band of T7-Syts I, II, T7-N-Gly-Syts VIII, IX (black columns), IC, IIC, T7-N-Gly-Syts VIIIC, and IXC (white columns). Immunoreactive bands in A were captured by Gel Print 2000i/VGA (Bio Image) and quantified by Basic Quantifier Software (version 1.0) (Bio Image). Columns indicate the proportion of endoglycosidase H-resistant bands (i.e. the proportion of proteins that were modified in the Golgi). Bars indicate the S.D. of three independent experiments.

Essential Role of the WHXL Motif in the C2B Domain Structure-- Finally, we investigated whether the WHXL motif is essential for the proper folding of the C2B domain biochemically by monitoring its sensitivity to proteolysis. If the above hypothesis were correct, mutation of the WHXL motif to AAXA would cause it to fail to form a stable C2 domain structure by disrupting the pairing of the -1 and -8 strands, and the mutant proteins should be more sensitive to proteinase than the wild-type proteins. When the GST-Syt IV-C2B423-425 proteins (including the WHXL motif) were treated with low concentrations of trypsin, they were rapidly cut into two pieces at the fusion site between GST (open arrowheads) and Syt IV-C2B (asterisk in Fig. 7, upper panel). The C2B423-425 proteins (including the WHXL motif) were highly resistant during 30 min of incubation at this concentration of trypsin, whereas the mutant C2B domains were degraded within 10 min. Thus, the WHXL motif was indeed found to be essential for the packed C2B domain structure.

View larger version (53K): [in this window] [in a new window]   Fig. 7.   Limited proteolysis of the C2B domain of synaptotagmin IV. After incubating 4 µg of GST-Syt IV-C2B423-425 (upper panel) and -C2B423-425(AAA) (lower panel) in 10 mM Tris-HCl, pH 7.5, and 100 mM NaCl with 1 ng/µl trypsin for 0-30 min at 25 °C, the reactions were stopped by addition of SDS sample buffer and boiling for 3 min. Samples were then analyzed by 12.5% SDS-PAGE and staining with Coomassie Brilliant Blue R-250. GST-Syt IV-C2B (closed arrowheads) was rapidly cut into two pieces at the fusion site between GST (open arrowheads) and Syt IV-C2B (asterisk). Note that the Syt IV-C2B423-425 proteins were highly resistant to 1 ng/µl trypsin, whereas the mutant Syt IV-C2B423-425(AAA) proteins were degraded within 10 min. The results shown are representative of two independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We previously found that deletion of 17 amino acids C-terminal of Syt IV induced granular structures and prevented the Golgi localization of Syt IV (41). In this study we showed by electron microscopy that this granular structure is crystalloid ER (36, 37) and analyzed the mechanism of its induction by expression of Syts I-XI lacking the conserved WHXL motif in the C terminus. Crystalloid ER was found to be induced by following processes: (a) Mutation or deletion of the conserved amino acids in the -1 or -8 strand (e.g. GE/DL/I in the -1 strand or WHXL in the -8 strand; see Fig. 3B) in the C2 domain destabilizes one of the four-stranded -sheets (i.e. anti-parallel pairing of the N-terminal -1 strand and the C-terminal -8 strand), preventing the mutant proteins from folding correctly. Misfolding of the C2B domain lacking the WHXL motif was confirmed by limited proteolysis (Fig. 7). Because anti-parallel pairing of other -sheets occurred between adjacent -3 and -4 or -6 and -7 strands (57), we speculate that mutations or deletions in the -sheets formed by the N-terminal -1 strand and the C-terminal -8 strand add a most striking effect to C2 domain structure. (b) The resulting malfolded proteins are correctly inserted into the ER (showing type I membrane topology) but then are not transported to the Golgi (Fig. 6) and accumulate in the ER. Accumulation of the malfolded proteins in the ER induces the expression of BiP, one of the ER stress proteins, which are known to be induced by accumulation of malfolded proteins and to be present in stable complexes in the malfolded proteins (61, 62). Consistent with this, Syt IVC proteins colocalized with BiP in the crystalloid ER (Fig. 1, E-G, arrows). Because the cytoplasmic domain of Syts I-XI lacking the WHXL motif (Syt-cytoC) was expressed in the cytosol, and its expression could not induce crystalloid ER (5, 43), malfolded proteins seem to be recognized only in the ER. (c) We previously proposed that crystalloid ER is formed by head-to-head association of malfolded molecules on apposed smooth ER membranes based on the results of an experiment using rat liver microsomal aldehyde dehydrogenase (37). It should be noted that the synaptotagmin family also shows Ca²⁺-independent oligomerization activity via around the spacer domain and/or fatty-acylated Cys cluster at the interface between the transmembrane and spacer domains, and disulfide bonding at the extracellular domain (17, 38-40, 59). Indeed, the expression of Syt Ispacer/C proteins, which lack oligomerization activity, could not induce crystalloid ER (Fig. 4). Therefore, it is most likely that the ERs in which Syt C proteins have accumulated associate with each other to form crystalloid ER as a result of the oligomerizing capacity of the Syt family.

Because the WHXL motif was first proposed to be an independent domain based on sequence comparisons with the C2A domain structure (4), many in vitro binding experiments investigating the C2B domain, including our own, have involved the use of recombinant C2B domains lacking the WHXL motif (i.e. -8 strand), as minimum C2B domains (25, 48, 63, 64, 66). Accordingly, some in vitro binding experiments on the C2B domain should be re-evaluated in terms of ligand binding specificity or ligand affinity. Indeed, we found that the C2B domain lacking the WHXL motif showed weaker Ca²⁺-dependent oligomerization activity in Syt VII than the wild-type protein (45) and found distinct inositol polyphosphate binding specificity in Syt II (24). Based on these findings, we suggest that differences in the length of the C terminus of the recombinant C2B domain, in addition to the impurity of recombinant proteins (67), are another important factor responsible for the contradictory results obtained in regard to the in vitro binding properties of the C2B domain (48, 65, 68-70).

Krasnov and Enikolopov (35) recently reported that mutation or deletion of the C terminus of Syt II prevented it from being transported to the tips of neurites where dense-core vesicles are abundant in nerve growth factor-differentiated PC12 cells, and they proposed that the WHXL motif is an active signal for targeting to the tips of neurites. However, our finding that the malfolded Syt C proteins lacking the WHXL motif are retained in the ER provides strong evidence against this idea. We suggest that Syt IIC proteins accumulate in the ER, are not transported to the Golgi, and, consequently, are not present in the dense-core vesicles derived from the TGN.

In conclusion, we have demonstrated that the WHXL motif functions as a protein interaction site (e.g. neurexins) as well as being important for proper folding of the C2B domain to stabilize anti-parallel -sheet formation between -1 and -8 strands. We propose that recombinant C2B domains that contain the entire C terminus should be used for in vitro binding experiments to avoid abnormal folding of the C2B domain, which is only evident in the ER of living cells.

	ACKNOWLEDGEMENTS

We thank Eiko Kanno, Chika Saegusa, and Yukie Ogata for technical assistance.

	FOOTNOTES

^* This work was supported by grants from the Science and Technology Agency to Japan (to K. M.) and Grants 13780624 from the Ministry of Education, Science, and Culture of Japan (to M. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Tel.: 81-48-467-9745; Fax: 81-48-467-9744; E-mail: mnfukuda@brain.riken.go.jp.

Published, JBC Papers in Press, August 30, 2001, DOI 10.1074/jbc.M106209200

	ABBREVIATIONS

The abbreviations used are: Syt(s), synaptotagmin(s); ER, endoplasmic reticulum; GST, glutathione S-transferase; HRP, horseradish peroxidase; PAGE, polyacrylamide gel electrophoresis; TGN, trans-Golgi network; BiP, immunoglobulin heavy chain binding protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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