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Originally published In Press as doi:10.1074/jbc.M404851200 on December 7, 2004

J. Biol. Chem., Vol. 280, Issue 8, 7262-7272, February 25, 2005
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Cellugyrin Induces Biogenesis of Synaptic-like Microvesicles in PC12 Cells*

Gabriel M. Belfort, Kyriaki Bakirtzi, and Konstantin V. Kandror{ddagger}

From the Boston University School of Medicine, Boston, Massachusetts 02118

Received for publication, April 30, 2004 , and in revised form, November 2, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The four-transmembrane domain proteins synaptophysin and synaptogyrin represent the major constituents of synaptic vesicles. Our previous studies in PC12 cells demonstrated that synaptogyrin or its nonneuronal paralog cellugyrin targets efficiently to synaptic-like microvesicles (SLMVs) and dramatically increases the synaptophysin content of SLMVs (Belfort, G. M., and Kandror, K. V. (2003) J. Biol. Chem. 278, 47971–47978). Here, we explored the mechanism of these phenomena and found that ectopic expression of cellugyrin increases the number of SLMVs in PC12 cells. Mutagenesis studies revealed that cellugyrin's hydrophilic cytoplasmic domains are not involved in vesicle biogenesis, whereas small conserved hydrophobic hairpins in the first luminal loop and the carboxyl terminus of cellugyrin were found to be critical for the formation of SLMVs. In addition, the length but not the primary sequence of the second luminal loop was essential for SLMV biogenesis. We suggest that changing the length of this loop similar to disruption of the short hydrophobic hairpins alters the position of the vicinal transmembrane domains that may be crucial for protein function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Understanding the mechanisms by which a cell sequesters specific proteins in unique subcellular compartments is fundamental to understanding how the cell maintains distinct organelles. For the past 25 years, the PC12 neuroendocrine cell line has provided a convenient system for studying the biogenesis of synaptic like microvesicles (SLMVs)1 (1). SLMVs were chosen as a model organelle, because, like neuronal synaptic vesicles, their small size (~50 nm) restricts the number of proteins present in each vesicle. Based on the estimates of 3 x 106 daltons of protein/synaptic vesicle and 50,000 daltons/average protein, ~60 proteins could be contained in a vesicle of this size (2).

Two main methods have been used to study the biogenesis of SLMVs. Several groups have studied cell-free or perforated cell vesicle budding systems in order to identify cytosolic factors critical for vesicle budding. Others studying specific proteins that target to SLMVs have identified regions in their primary amino acid sequences that are required for targeting to that compartment.

Two main pathways of SLMV biogenesis have emerged from in vitro studies. First, vesicles can bud from endosomes in a brefeldin A-sensitive mechanism that requires the AP3 adaptor protein and the ARF1 and Rab4 GTPases (3–6). By the second pathway, SLMVs can bud from the plasma membrane in a brefeldin A-insensitive mechanism that involves the AP2 adapter, clathrin, and dynamin (7). Further work with perforated PC12 cells demonstrated that endophilin I, a dynamin-binding protein, facilitates SLMV budding, probably by modifying the membrane lipid composition (8).

Two categories of proteins are present in SLMVs in PC12 cells: neuronal synaptic vesicle proteins and other proteins that are not found in brain synaptic vesicles. Of those proteins belonging to the former category, synaptophysin, synaptobrevin/VAMPII, the vesicular acetylcholine transporter, and synaptotagmin I have all been at least partially analyzed for SLMV targeting sequences (9–13).

The group of nonsynaptic vesicle proteins localized in SLMVs can be further divided into the nonneuronal paralogs of synaptic vesicle proteins (cellubrevin, cellugyrin, and pantophysin) (14–16) and those proteins that are unrelated to synaptic vesicle proteins (tyrosinase and P-selectin) (13, 17). These studies, which examined targeting motifs, failed to identify a universal sequence applicable to all SLMV proteins. The one commonality among all of the targeting motifs identified so far is that they are all located in cytoplasmic domains. The question then arises as to what mechanisms maintain the structural integrity of SLMVs, such that, despite the complexity of their trafficking pathways in the cells and apparent heterogeneity of targeting signals, these vesicles maintain uniform and specific protein composition? Undoubtedly, adaptor proteins provide a certain level of specificity upon vesicle budding from the donor membranes. However, given the fact that at least two adaptors, AP-2 and AP-3, with different binding specificities, are involved in the formation of vesicles with the same protein composition from different compartments, we speculate that additional factors should participate in the formation of SLMVs.

Cellugyrin (also referred to as synaptogyrin 2) is the nonneuronal paralog of the synaptic vesicle protein, synaptogyrin 1. Both cellugyrin and synaptogyrin 1 target to SLMVs in PC12 cells, and both do so with a much higher efficiency than any other protein tested so far (15). Increasing the level of cellugyrin or synaptogyrin 1 in PC12 cells also causes a concomitant increase of endogenous synaptophysin in SLMVs.

Here, we report that cellugyrin expression is sufficient to induce the biogenesis of SLMVs, as measured by two independent methods. Furthermore, despite the finding that cellugyrin and synaptogyrin are capable of targeting very efficiently to microvesicles in all cell types tested, the capacity to facilitate targeting of synaptophysin to microvesicles was not replicated in a nonneuronal cell line (COS7 cells). These results suggest that the capacity of cellugyrin to facilitate the targeting of synaptophysin is not an artifact of overexpression but rather represents an epiphenomenon of cellugyrin targeting to microvesicles that requires cell type-specific factors. In support of this latter hypothesis, molecular dissection of cellugyrin via recombinant DNA techniques revealed that the targeting of cellugyrin mutants to SLMVs can be uncoupled from cellugyrin's effects on synaptophysin targeting. These studies demonstrated that conserved hydrophobic sequences in the carboxyl-terminal domain and the first luminal loop, in addition to the lengths, but not the primary sequences, of the second luminal loop and the cytoplasmic loop are key determinants for efficient SLMV targeting. Furthermore, inefficiencies in the targeting of these mutants to SLMVs yielded an even more significant decrease in the facilitation of synaptophysin targeting. We hypothesize that the highly efficient targeting of cellugyrin and synaptogyrin 1 to SLMVs and their ability to facilitate the targeting of synaptophysin is dependent on their capacity to increase the number of SLMVs and that this function is mediated by the regions identified in our targeting studies.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Plasmid pCMV5-p38, which expresses rat synaptophysin, pCMV-Cgyr, which expresses rat cellugyrin, and pCMV5-p29, which expresses rat synaptogyrin 1, were all kind gifts from Dr. T. C. Südhof (University of Texas Southwestern). Plasmid pBABE-mycGlut4, which contains a 110-amino acid 7x Myc tag, was the kind gift of Jun Shi (Boston University School of Medicine). Plasmids pcDNA3.1/myc-His(+) and pcDNA3.1His(+) were purchased from Invitrogen. Anti-cellugyrin monoclonal antibody was purchased from BD Biosciences. Anti-HisG monoclonal antibody, which recognizes the amino-terminal His tag, was purchased from Invitrogen. Anti-synaptophysin monoclonal antibody was purchased from Chemicon. Anti-VAMPII/synaptobrevin monoclonal antibody and anti-synaptogyrin polyclonal antibody was purchased from Synaptic Systems. Anti-Myc monoclonal and polyclonal antibodies were purchased from Cell Signaling. Anti-calnexin polyclonal antibody was purchased from Stressgen. Anti-tubulin monoclonal antibody was purchased from Sigma.

Recombinant DNA Constructs—Plasmid pHis-cellugyrin, encoding rat cellugyrin with an N-terminal His tag, was reported previously (15). Table I contains information on how the plasmids used in these studies were constructed.


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  		TABLE I
Recombinant DNA constructs

 
Cell Culture and Transfections—The pheochromocytoma cell line, PC12, was grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 50 µg/ml gentamicin, 100 units/ml penicillin, and 100 µg/ml streptomycin in 5% CO2 at 37 °C. COS7 cells, a monkey kidney cell line, were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 µg/ml gentamicin, 100 units/ml penicillin, and 100 µg/ml streptomycin in 5% CO2 at 37 °C. Transfections were performed with Lipofectamine2000 reagent (Invitrogen) according to the manufacturer's instructions. Optimal transfections were obtained with 5 µg of DNA/10-cm plate and a Lipofectamine2000 reagent to plasmid ratio of 3 µl/µg of DNA. The efficiency of transfection was over 90%. Stably transfected cell lines were selected by culturing transiently transfected cells in the presence of G418 for 30 days.

Vesicle Isolation and Fractionation of PC12 Cells—Confluent 15- or 10-cm plates were washed once with PBS. Cells were removed from the plates with cell dissociation medium (Sigma) for PC12 cells or trypsin/EDTA (Invitrogen) for COS7. Cells in suspension were pelleted at 400 x g for 10 min. Pelleted cells were resuspended in Buffer A (150 mM NaCl, 10 mM HEPES, pH 7.4, 1 mM EGTA, 0.1 mM MgCl2) with protease inhibitors (1 µM aprotinin, 5 mM benzamidine, 2 µM leupeptin, 1 µM pepstatin, 1 mM phenylmethylsulfonyl fluoride). Resuspended cells were homogenized with 11 strokes through a ball bearing cell cracker (European Molecular Biology Laboratory) and centrifuged at 1000 x g for 5 min to generate a postnuclear supernatant (PNS). A high speed supernatant (S2) that contains only microvesicles and cytosolic proteins was generated by centrifugation of the PNS at 27,000 x g (15,000 rpm) for 35 min in a Ti42.2 rotor (Beckman) (18). Density gradient centrifugation was performed by layering ~1 mg of S2, adjusted to 230 µl, onto a 4.6-ml 10–50% sucrose gradient in Buffer A (w/v). Density gradients were centrifuged at 280,000 x g (48,000 rpm) in an SW55 rotor (Beckman) for 16 h. Fractions were collected from the bottom via a peristaltic pump. Vesicle decoration/shift analysis was performed by incubating 1 mg of S2 with 2.5 µg of purified nonspecific mouse IgG or anti-synaptophysin monoclonal antibodies, together with 2 µg of Nanogold-conjugated goat anti-mouse Fab fragments (Nanoprobes). This mixture was rotated at 4 °C for 2 h. The total volume of decorated vesicles (always adjusted to 230 µl) was then loaded on a 4.6-ml linear 10–30% sucrose gradient in Buffer A (w/v). Gradients were centrifuged at 280,000 x g (48,000 rpm) in an SW55 rotor (Beckman) for 1 h and 15 min. Fractions were collected from the bottom via a peristaltic pump.

Vesicle Isolation and Fractionation of COS7 Cells—Confluent 15- or 10-cm plates were washed once with PBS. Cells were removed from the plates with trypsin/EDTA (Invitrogen). Cells in suspension were pelleted at 400 x g for 10 min. Pelleted cells were resuspended in Buffer A with protease inhibitors. Resuspended cells were homogenized with 11 strokes through a ball bearing cell cracker (European Molecular Biology Laboratory) and centrifuged at 16,000 x g for 30 min. The pellet (16,000 x g pellet) represents total expression, and the supernatant (16,000 x g supernatant) represents the microvesicular fraction. The 16,000 x g pellet was extracted with a 1% solution of Triton X-100 in Buffer A. This isolation method was tested in tandem with the PNS/S2 isolation method (see above) in PC12 cells and found to be functionally equivalent.

Radioactive Labeling and Organelle Immunoisolation—Whole cell lipids were labeled as previously reported (19). Briefly, cells were incubated overnight with 8 µCi of [1-14C]acetic acid, sodium salt (40–60 mCi/mmol) (PerkinElmer Life Sciences). A PNS was prepared and then assayed for protein concentration. PNS from empty vector- and pHis-cellugyrin-transfected cells were adjusted to the same protein concentration. A high speed supernatant was prepared (S2). Goat anti-mouse magnetic beads (Dynal) were precoated for 2 h with 2 µg of nonspecific IgG or synaptophysin antibody and washed with Buffer A. Equal quantities of IgG or synaptophysin-coated beads were mixed overnight in S2 from empty vector- or synaptophysin-transfected cells. Unbound material, referred to here as postabsorptive supernatants (PAS) was removed. The beads were washed three times with Buffer A. Triton elution (TE), with 1% Triton X-100 in Buffer A, was used to elute the lipids from the beads. SDS elution (SE), performed with Laemmli sample buffer, was used to remove the bound antibody (anti-synaptophysin) and its ligand (synaptophysin) from the bead (20). Radioactivity of the culture media at time 0 and 24 h, PNS, S2, PAS, TE, and SE was quantified in a 1217 cintillation counter (LKB Wallace). In addition, PAS, TE, and SE were subjected to Western blot analysis.

Immunofluorescence—PC12 cells transfected with pMyc/His-CR1–177-SCR67–76, which codes for rat cellugyrin with a truncated carboxyl terminus, a scrambled first luminal loop, and a carboxyl-terminal Myc/His tag, were lifted and grown on coverslips coated with poly-L-lysine overnight. Cells were then fixed with 4% paraformaldehyde in PBS for 30 min, washed with PBS, permeabilized with 0.2% Triton X-100 for 5 min, blocked with 4% donkey serum, and probed with either monoclonal anti-Myc and polyclonal anti-calnexin or polyclonal anti-Myc and monoclonal anti-synaptophysin antibodies followed by Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch) and Alexa 488-conjugated donkey anti-mouse IgG (Molecular Probes, Inc., Eugene, OR). Incubation with primary and secondary antibodies lasted for 60 min at room temperature and was followed by six quick rinses with PBS. Next, nuclei were stained with 4',6-diamidino-2-phenylindole dihydrochloride according to the manufacturer's instructions (Molecular Probes). The SlowFade-Light Antifade kit (Molecular Probes) was used for mounting coverslips onto slides. Staining was examined by fluorescence microscopy (Axiovert 200M; Zeiss).

Gel Electrophoresis and Immunoblotting—Protein samples were separated by SDS-PAGE according to Laemmli and transferred to polyvinylidene difluoride membranes in 25 mM Tris, 192 mM glycine (20). Following transfer, the membrane was blocked with 10% nonfat dry milk in PBST for 1 h at 25 °C and probed with specific antibodies overnight. The following day membranes were washed three times with phosphate-buffered saline with 0.05% Tween and incubated with horseradish peroxidase-labeled secondary antibody for 1 h at room temperature. After three more washes, the membranes were incubated in ECL reagent (PerkinElmer Life Sciences) for 1 min and then exposed to an Eastman Kodak Co. 440 image station. Data analysis was performed with Kodak 1D image analysis software.

Hydropathy Plot Generation—The hydropathy plot of rat cellugyrin was generated by the Kyte-Doolittle method available on the Biology Workbench (available on the World Wide Web at workbench.sdsc. edu/) (21).

Statistical Analysis—Student's unpaired two-tailed t test was used to evaluate the statistical significance of the differences in targeting efficiencies. Targeting efficiency is defined as the amount of a specific protein in the S2 fraction normalized to the total amount of the same protein in the PNS. A paired t test was used to evaluate the statistical significance of the difference between synaptophysin-attributable radioactive counts in the Triton X-100 extract from empty vector and pHis-cellugyrin-transfected cells. Synaptophysin-attributable counts are defined as the counts precipitated with synaptophysin antibody minus the counts precipitated with nonspecific IgG.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cellugyrin Increases the Number of SLMVs in the Cell—In order to further characterize the biological role of cellugyrin, we explored the impact of exogenous cellugyrin expression on the physical characteristics of the SLMV. In a previous publication (15), we showed that increasing the cellugyrin levels in the SLMV had no impact on vesicle size, since sedimentation in glycerol velocity gradients was unchanged. Now we asked whether the vesicles changed in buoyant density, hypothesizing that an increase in protein per vesicle should increase the buoyant density of the vesicles (Fig. 1). High speed supernatants (S2) from empty vector and pHis-cellugyrin-transfected cells were loaded on continuous 10–50% sucrose gradients and centrifuged for 16 h at 280,000 x g. The peak of cellugyrin and synaptophysin in both empty vector and His-cellugyrin-transfected cells is between fractions 11 and 15 (Fig. 1). Note that both endogenous and transfected cellugyrin signals are detectable in the lower cellugyrin panel. These experiments reveal that the vesicle pool from cellugyrin-transfected cells are the same buoyant density as those from empty vector-transfected cells.



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  		FIG. 1.
Ectopic cellugyrin expression does not alter buoyant density of SLMV. 15-cm plates of PC12 cells were transfected with 10 µg of either pcDNA3.1His(+) (empty vector) or pHis-cellugyrin. Two days after transfection, cells were homogenized, and a low speed supernatant was obtained (PNS). All of the PNS was then used to generate a high speed supernatant (S2). Equal protein of S2 (1 mg) from empty vector- and pHis-cellugyrin-transfected cells was loaded onto continuous 10–50% sucrose gradients. After centrifugation at 280,000 x g (48,000 rpm) in an SW55 rotor (Beckman) for 16 h, fractions were collected from the bottom in 200-µl aliquots. Equal volumes of odd fractions were mixed with loading buffer and separated by SDS-PAGE followed by transfer to polyvinylidene difluoride and probing with monoclonal antibodies against cellugyrin and synaptophysin. The numbers represent gradient fractions. WB, Western blot.

 
Given that the vesicles are the same size and buoyant density, we hypothesized that cellugyrin increases the number of vesicles per cell and that these new vesicles accommodate the increased accumulation of proteins. We tested this hypothesis using two independent approaches. First we used a method developed in our laboratory called antibody decoration/shift analysis. This method involves the addition of an excess of a single monoclonal antibody to a high speed supernatant (S2) containing the SLMVs. During the incubation period, the antibody binds to the cytoplasmic epitope of the target protein in a 1:1 stoichiometric ratio. Excess Nanogold-conjugated goat anti-mouse Fab fragments are included in the incubation to add more mass to each antibody. Next, the decorated vesicles are loaded on to a linear 10–30% sucrose gradient and centrifuged (see "Experimental Procedures"). Vesicles with a high copy number of the target protein migrate farther than vesicles with a lower copy number, because they are bound by more specific antibodies and in turn more Nanogold-labeled Fab fragments. In Fig. 2, we compared the shift of vesicles from empty vector- and cellugyrin-transfected cells using synaptophysin as the shifting antibody and nonspecific purified mouse IgG as the control. In the presence of nonspecific IgG, vesicles from both empty vector- and His-cellugyrin-transfected cells sediment with a peak between fractions 15 and 17. Anti-synaptophysin antibody shifts both peaks to between fractions 1 and 13. These data suggest that vesicles from cellugyrin-over-expressing cells and empty vector controls contain the same number of synaptophysin molecules. This implies that the increase in synaptophysin seen in the high speed supernatant (S2) is in fact explained by a greater number of vesicles.



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  		FIG. 2.
Ectopic cellugyrin expression increases the synaptophysin content of the SLMV pool but does not increase the copy number of synaptophysin per vesicle. 15-cm plates of PC12 cells were transfected with 10 µg of either pcDNA3.1His(+) (empty vector) or pHis-cellugyrin. Two days after transfection, cells were homogenized, and a low speed supernatant was obtained (PNS). All of the PNS was then used to generate a high speed supernatant (S2). Equal amounts of protein of S2 (1 mg) from empty vector- and pHis-cellugyrin-transfected cells were incubated at 4 °C for 2 h with 2 µg of Nanogold-conjugated anti-mouse Fab fragments and 2.5 µg of nonspecific mouse IgG or anti-synaptophysin monoclonal antibodies (Ab). Decorated S2 was then fractionated in a 10–30% linear sucrose gradient. After centrifugation at 280,000 x g (48,000 rpm) in an SW55 rotor (Beckman) for 1 h and 15 min, fractions were collected from the bottom in 200-µl aliquots. The bottom of the centrifuge tube (pellet) was thoroughly washed with 200 µl of Buffer A. Equal volumes of odd fractions and pellet were mixed with loading buffer and separated by SDS-PAGE followed by transfer to polyvinylidene difluoride and probing with antibodies against synaptophysin and cellugyrin. Proteins shown in the same rows were transferred to the same membrane and developed simultaneously, so these signals are directly comparable. Staining in the top gradient fractions represents a nonspecific signal. WB, Western blot primary antibody; P, pellet; numbers, gradient fractions.

 
In order to verify the result obtained by antibody decoration/shift analysis, radioactive lipid labeling experiments were performed (Fig. 3, A and B). Cells transiently (Fig. 3A) or stably (Fig. 3B) transfected with either empty vector or His-cellugyrin were incubated in [1-14C]acetate overnight. A PNS was isolated, followed by adjustment of the protein concentrations of the different PNS samples so that they were equal. Then a high speed supernatant (S2) was obtained and split into two aliquots, one for immunoadsorption with anti-synaptophysin-coated goat antimouse magnetic beads and the other for mock immunoadsorption with nonspecific IgG-coated beads. The precipitated lipids along with vesicular proteins were eluted with 1% Triton X-100 (TE). Antibody and ligand were then eluted with SDS-containing sample buffer (SE). Liquid scintillation counting of the TE samples revealed ~2.5-fold increase in the lipid content in these vesicles (p < 0.03) (Fig. 3, A and B). Given that the vesicles are the same size and density, this strongly suggests that expression of cellugyrin leads to an increase in the number of SLMVs. Fig. 3C demonstrates a Western blot analysis of fractions obtained during organelle immunoisolation.



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  		FIG. 3.
Cellugyrin increases the number of SLMVs in PC12 cells. A, 10-cm plates of PC12 cells were transfected with 4 µg of either pcDNA3.1His(+) (white bar) or pHis-cellugyrin (black bar). The day after transfection, growth medium was supplemented with 8 µCi of [1-14C]acetic acid, sodium salt (40–60 mCi/mmol) (PerkinElmer Life Sciences). Two days after transfection, cells were homogenized, and a low speed supernatant was obtained (PNS). PNS from empty vector- and cellugyrin-transfected cells was ad-justed to the same protein concentration. All of the PNS was then used to generate a high speed supernatant (S2). Equal volumes of S2 were then incubated overnight with goat anti-mouse magnetic beads (Dynal) precoated with nonspecific mouse IgG or anti-synaptophysin monoclonal antibody. The PAS was removed, and the beads were washed three times with Buffer A. Vesicular lipids were eluted by incubating the beads at 4 °C for 30 min in 1% Triton X-100 in Buffer A (TE). Half of the Triton eluate was submitted for liquid scintillation counting. The N values refer to the number of experiments. *, statistical significance (p < 0.03). After solubilization of the vesicular membrane in Triton X-100 (TX), specific antibody and ligand were eluted with SDS sample buffer (SE) by vortexing at 25 °C. B, experiments were performed as described for A with stably transfected cells. C shows a representative Western blot (WB) of equal volume aliquots of the PAS, Triton eluate (TE), and SDS eluate (SE). Nonspecific signals from the IgG light chain (25 kDa) in SDS eluates are boxed.

 
Cellugyrin's Effect on Synaptophysin Targeting Is Specific for PC12 Cells—Cellugyrin is a marker for ubiquitous microvesicles present in all cell types tested, including the COS7 monkey kidney cell line (15). In order to explore whether or not gyrins can exert their effects in nonneuronal cells, we co-transfected His-cellugyrin (or His-synaptogyrin) with His-synaptophysin in COS7 cells (Fig. 4). Note that synaptophysin ectopically expressed in COS7 cells represents a doublet, which is consistent with earlier results of Linstedt and Kelly (22). Total expression levels per microgram of transfected DNA of all three proteins were similar to one another in COS7 as well as in PC12 cells. In agreement with previously published results (15), a significant fraction of ectopically expressed cellugyrin and synaptogyrin were found in the fraction of light vesicles recovered in the 16,000 x g supernatant of both COS7 and PC12 cells, suggesting that proteins of the gyrin family can facilitate formation of microvesicles in various different cells. Synaptophysin alone was not targeted efficiently to microvesicles in either cell type. In addition, cellugyrin and synaptogyrin did not facilitate the targeting of synaptophysin to the microvesicular compartment in COS7 cells, as they do in PC12 cells. These results argue against the possibility that the facilitation of synaptophysin targeting by either cellugyrin or synaptogyrin 1 represents an artifact of protein overexpression. Instead, these data suggest that synaptophysin targeting to the newly formed compartment requires a factor or factors that are only expressed in certain cell types.



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  		FIG. 4.
Cellugyrin and synaptogyrin facilitate targeting of synaptophysin to microvesicles in PC12, but not in COS7 cells. 10-cm plates of COS7 cells (A) or PC12 cells (B) cells were transfected with 2 µg of pHis-synaptophysin and either 2 µg of pcDNA3.1His(+) (empty vector), pHis-cellugyrin, or pHis-synaptogyrin. Two days after transfection, cells were homogenized and centrifuged at 16,000 x g for 30 min. Equal quantities (50 µg) of the Triton X-100 (TX)-extracted 16,000 x g pellet and 16,000 x g supernatant were separated by SDS-PAGE followed by Western blot analysis (WB). All bands in both panels were visualized with the monoclonal anti-HisG antibody (Invitrogen).

 
Mapping of Functional Domains of Cellugyrin—A comprehensive analysis of the rat cellugyrin protein was undertaken using recombinant DNA technology to identify domains responsible for the formation of SLMVs as well as cellugyrin's effect on synaptophysin targeting in PC12 cells (15). We based our approach to the molecular dissection of this 234-amino acid protein (cellugyrin accession number AAB96666 [GenBank] on the fourtransmembrane domain (TM1, -2, -3, and -4) structure suggested by Südhof and colleagues, which implies that both the amino and carboxyl termini are located in the cytosol with one cytosolic loop (between TM2 and TM3) and two luminal loops (between TM1 and TM2 and between TM3 and TM4) (23).

In targeting studies, the level of a given protein in a postnuclear extract (1000 g for 5 min; referred to as PNS) was taken to represent the total level of expression, whereas the level of the protein in a high speed supernatant (27,000 x g for 35 min; referred to as S2) was taken as a measure of the protein in the microvesicular pool (18). All mutants analyzed statistically were present at total (PNS) levels greater than 80% of wild type cellugyrin. We divided our analysis of the primary amino acid sequence into three putative control regions: 1) termini (cytoplasmic amino and carboxyl termini), 2) loop regions (luminal loop 1 and 2 and the cytoplasmic loop), and 3) transmembrane domains.

Previous studies performed with rat synaptogyrin 1 and the C. elegans synaptogyrin homolog showed that the carboxyl termini of those proteins were required for reaching nerve termini in cultured hippocampal neurons and intact worms (24). Therefore, we decided to focus our initial efforts on the C terminus of cellugyrin. Deletion of the C terminus by substituting Gln178 with a stop codon yielded an unstable construct (data not shown). However, we were able to recover stability by placing a Myc/His tag after Tyr177 (construct name pMyc/HisCR1–177) (Figs. 5 and 8). In comparison with the wild type protein with the same tag, this construct showed a 44% decrease in mean vesicle targeting/formation efficiency (p < 0.01) and a 59% decrease in its effect on mean synaptophysin targeting efficiency (p < 0.01).



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  		FIG. 5.
The carboxyl-terminal domain of cellugyrin is required for targeting to the microvesicle membrane fraction. 10-cm plates of PC12 cells were transfected with 4 µg of pcDNA3.1/myc-His(+) (empty vector), pMyc/His-cellugyrin, or pMyc/His-CR1–177. Two days after transfection, cells were homogenized, and a low speed supernatant was obtained (PNS). Half of the PNS was then used to generate a high speed supernatant (S2). Equal quantities (50 µg) of PNS and S2, corresponding to roughly equal amounts of starting material, were separated by SDS-PAGE followed by Western blot analysis. Western blot primary antibodies were anti-cellugyrin monoclonal (BD Transduction Laboratories) and anti-synaptophysin monoclonal (Chemicon). The hatched area in the schematic diagram identifies the region of cellugyrin addressed in this figure.

 



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  		FIG. 8.
Quantification of mutant function. SLMV targeting efficiency is defined as the S2 (microvesicle) signal of a given protein divided by the PNS (total) signal of the same protein as determined by Western blot densitometry. Targeting efficiency for all cellugyrin mutants was normalized to wild-type cellugyrin with the same tag. Targeting efficiency of synaptophysin for cells transfected with various cellugyrin mutants was normalized to synaptophysin targeting efficiency of wild-type cellugyrin with the same tag. Asterisks mark a statistically significant difference in normalized targeting efficiency in comparison with wild type cellugyrin with the same tag (see "Results" for p values). N values indicate the number of experiments in which targeting efficiency was determined for that construct.

 
Having identified the carboxyl terminus as a domain required for cellugyrin function, we made a series of C-terminal truncations, leaving a sufficient number of amino acids after TM4 so that the protein was stable. Substitution of amino acids Ile188, Phe198, Ala208, Tyr213, and Phe218 by stop codons did not yield a construct that differed in intracellular localization from the wild type protein (data not shown). Thus, we suggest that amino acids 178–187 are essential for efficient SLMV formation and altering synaptophysin targeting. This region of the carboxyl terminus is clearly hydrophobic and is conserved in all known synaptogyrin paralogs (synaptogyrin 1, 2, and 3) and orthologs (synaptogyrin from mouse, rat, human, and Caenorhabditis elegans) (Fig. 6B).



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  		FIG. 6.
The first luminal loop of cellugyrin contains a conserved hydrophobic region required for targeting to the microvesicle membrane fraction. A, 10-cm plates of PC12 cells were transfected with 4 µg of pcDNA3.1His(+) (empty vector), pHis-cellugyrin, pHis-SCR67–76, pHis-67–69toA, pHis-70–73toA, or pHis-74–76toA (two plasmid clones). Two days after transfection, cells were homogenized, and a low speed supernatant was obtained (PNS). Half of the PNS was then used to generate a high speed supernatant (S2). Equal quantities (50 µg) of PNS and S2, corresponding to roughly equal amounts of starting material, were separated by SDS-PAGE followed by Western blot analysis. The hatched area in the schematic diagram identifies the region of cellugyrin addressed in this panel. B, a hydropathy plot of rat cellugyrin exhibiting the conserved hydrophobic region (shaded area).

 
Analysis of the amino terminus (amino acids 1–40) of the protein revealed that the first 26 amino acids can be truncated without loss of function (data not shown). Complete truncation of the amino terminus or deletion of amino acids 26–39, which reside next to TM1, resulted in degradation of the protein (data not shown). Flexibility in regard to the first 26 amino acids is consistent with the fact that wild type rat cellugyrin is functional despite 10 additional amino-terminal residues in comparison with human and mouse cellugyrin.

We next studied the luminal and cytoplasmic loops, by scrambling the order of a 9- or 10-amino acid region in each loop, choosing the regions farthest away from predicted transmembrane domains. All three resulting constructs were stable with varying levels of impact on cellugyrin function. The first luminal loop, amino acids 67–76, was mutated from the wild type sequence (67LHCVFNRNED76) to SERDHVNLFN (construct name pHis-SCR67–76) (Figs. 6A and 8). In comparison with His-tagged wild type cellugyrin, the His-SCR67–76 protein had a 41% decrease in mean vesicle formation/targeting efficiency (p < 0.001) and a 77% decrease in its effect on mean synaptophysin targeting efficiency (p < 0.001).

An alanine scan of amino acids 67–76 was initiated. Three constructs were made: 1) pHis-67–69toA (in which amino acids 67–69 were mutated to alanine); 2) pHis-70–73toA; and 3) pHis-74–76toA. The His-67–69toA protein was decreased in both PNS and S2, so statistical analysis was not performed. The His-70–73toA protein was degraded even more completely, but the His-74–76toA protein behaved indistinguishably from wild type cellugyrin (Fig. 6A). This narrowed the functional area of this region down to amino acids 67–73, with last four amino acids being more crucial than the first three in terms of stability. Finally, isolated substitution of asparagine 72 or cysteine 69 with alanine did not alter the behavior of the protein (data not shown). Together, these data suggest that hydrophobic amino acids in this region are more critical for cellugyrin functions than hydrophilic amino acids. This is consistent with the fact that synaptogyrin 1, 2, and 3 from mice, rats, and humans and the C. elegans homolog of synaptogyrin (SNG1) all have a predicted short hydrophobic region that intervenes between TM1 and TM2 (Fig. 6B).

Scrambling of cellugyrin's second luminal loop (142ATKPD-DVLVG151) to KVVDLTAGPD (construct name pHis-SCR142–151) had no impact on the function of the protein (Fig. 7A). In order to further explore the flexibility of this region, we inserted a 13-amino acid Myc tag between glycine 149 and proline 150 (construct name pHis-MycinSCR142–151). The His-MycinSCR142–151 protein was stable, but compared with wild type cellugyrin, it exhibited a 46% decrease in mean vesicle formation/targeting efficiency (p < 0.01) and a 77% decrease in its effect on mean synaptophysin targeting efficiency (p < 0.01) (Figs. 7A and 8).



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  		FIG. 7.
The lengths, but not the primary sequences, of the cytoplasmic and second luminal loops are critical for targeting to the microvesicle membrane fraction. A, 10-cm plates of PC12 cells were transfected with 4 µg of pcDNA3.1His(+) (empty vector), pHis-cellugyrin, pHis-SCR142–151, pHis-MycinSCR142–151, or pHis-7MycinSCR142–151. Two days after transfection, cells were homogenized, and a low speed supernatant was obtained (PNS). Half of the PNS was then used to generate a high speed supernatant (S2). Equal quantities (50 µg) of PNS and S2, corresponding to roughly equal amounts of starting material, were separated by SDS-PAGE followed by Western blot analysis. The hatched area in the schematic diagram identifies the region of cellugyrin addressed in this panel. B, 10-cm plates of PC12 cells were transfected with 4 µg of pHis-cellugyrin, pHis-SCR104–112, pHis-MycinSCR104–112, or pHis-SCR67–69 (negative control). Two days after transfection, cells were homogenized, and a low speed supernatant was obtained (PNS). Half of the PNS was then used to generate a high speed supernatant (S2). Equal quantities (50 µg) of PNS and S2, corresponding to roughly equal amounts of starting material, were separated by SDS-PAGE followed by Western blot analysis. The hatched area in the schematic diagram identifies the region of cellugyrin addressed in this panel.

 
Cellugyrin's single cytoplasmic loop (104QISNATDRK112) was scrambled to DIQRSAKTN (construct name pHis-SCR104–112) without impacting the function of the protein. Also, there was no detectable change in function when a 10-amino acid Myc tag was inserted into the cytoplasmic loop (Fig. 7B), in contrast with the single Myc tag inserted in the second luminal loop.

In the following experiment, the C-terminal deletion (pMyc/His-CR1–177) was combined with the first luminal loop scramble (pHis-SCR67–76) to create a double mutant (construct name pMyc/His-CR1–177-SCR67–76). This latter construct was partially degraded but was still readily detectable on Western blots (Fig. 9). Targeting of the double mutant to the SLMV was completely eliminated, since it was undetectable in the S2 fraction, even when the blot was overexposed. In order to confirm this result, we performed immunocytochemistry on PC12 cells transfected with pMyc/His-CR1–177-SCR67–76 (Fig. 10). Despite the low levels of expression seen by Western blot, cells contained detectable levels of the protein. Double staining with anti-Myc and anti-calnexin (an ER marker) antibodies revealed that the double mutant exits the ER (Fig. 10A). On the other hand, cells probed with anti-Myc and anti-synaptophysin anti-bodies show that the double mutant does not co-localize with an SLMV marker (Fig. 10B), confirming the results obtained by Western blot. Note that wild-type cellugyrin co-localized perfectly with synaptophysin in previous immunocytochemistry studies (15). Together these data suggest that the double mutant folds correctly and exits the ER and then cannot target to SLMVs, which leads to degradation of the protein.



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  		FIG. 9.
A double mutant with a scrambled first luminal loop and a deleted carboxyl terminus does not target to SLMVs. 10-cm plates of PC12 cells were transfected with 4 µg of pcDNA3.1/myc-His(+) (empty vector), pMyc/His-cellugyrin, or pMyc/His-CR1–177-SCR67–76. Two days after transfection, cells were homogenized, and a low speed supernatant was obtained (PNS). Half of the PNS was then used to generate a high speed supernatant (S2). Equal quantities (50 µg) of PNS and S2, corresponding to roughly equal amounts of starting material, were separated by SDS-PAGE followed by Western blot analysis. The hatched areas in the schematic diagram identify the regions of cellugyrin addressed in this figure.

 



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  		FIG. 10.
The double mutant (pMyc/His-CR1–177-SCR67–76) is not sequestered in the ER and does not colocalize with synaptophysin. PC12 cells ectopically expressing His-CR1–177-SCR67–76 were grown on glass coverslips and fixed with 4% paraformaldehyde in PBS. Cells were then permeabilized by incubation in 0.2% Triton X-100 in PBS for 5 min. Cells were then washed three times with PBS and blocked for 1 h at room temperature in blocking solution (5% donkey serum, 5% BSA in PBS). Cells were then incubated for 1 h at room temperature in anti-Myc monoclonal (1:1000 dilution) and anti-calnexin polyclonal (1: 1000 dilution) made in blocking solution (A) or anti-Myc polyclonal (1:1000 dilution) and anti-synaptophysin monoclonal (1:1000 dilution) in blocking solution (B). Then all coverslips were washed six times with PBS and incubated for 1 h at room temperature in Alexa 488-conjugated donkey anti-mouse (1:500 dilution) and Cy3-conjugated donkey anti-rabbit (1:1000 dilution) made in blocking solution. The cells were rinsed six times with PBS, and the nuclei were stained with 4',6-diamidino-2-phenylindole dihydrochloride by the method recommended by the manufacturer (Molecular Probes). Coverslips were mounted on glass slides with the SlowFade-Light Anti-fade kit (Molecular Probes). Cells were visualized on a Zeiss Axiovert 200M fluorescence microscope. Bars, 4 µM.

 
Finally, we sequentially substituted half of each putative transmembrane domain with the artificial peptide LAAAAVLAVV. Hypothetical analysis (performed on-line with Biology Workbench) of all theoretical constructs suggested that this peptide substitute would have wild-type characteristics of helicity and hydrophobicity. In each case, the peptide was predicted to be part of a transmembrane domain. Seven of the eight resulting polypeptides were completely degraded. The one stable protein, substitution of the luminal half of TM2, was indistinguishable from wild-type cellugyrin (data not shown). Nonetheless, the stability of this construct suggests that the predictions of hydrophobicity and helicity were accurate and that it is the specific sequences of the other transmembrane domain regions that are required for protein stability.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Upon ectopic expression of cellugyrin or synaptogyrin 1 in PC12 cells, a significant fraction of both proteins is targeted to SLMVs. Simultaneously, both proteins are capable of increasing the synaptophysin content in SLMVs (15). Given that synaptogyrin 1 and synaptophysin are reported to account for 10% of the total protein in synaptic vesicles, we wondered how the SLMVs adapted to this massive increase in cargo. Velocity gradient centrifugation revealed that ectopic expression of cellugyrin does not alter the average vesicle size in the SLMV pool (15). Also, the buoyant density of the vesicles from cells transfected with cellugyrin is similar to the buoyant density of vesicles from control cells (Fig. 1). Furthermore, using antibody decoration/shift analysis, we found that the average number of synaptophysin molecules per vesicle in cellugyrin-overexpressing cells is the same as in empty vector-transfected cells (Fig. 2), a paradox that can only be reconciled by concluding that there are more vesicles synthesized.

The data from the lipid radiolabeling experiments provide strong evidence that such an explanation is correct. In these studies (Fig. 3), vesicle immunoisolation with anti-synaptophysin antibody revealed at least a 2.4-fold increase in the lipid content of SLMVs from cellugyrin-transfected cells compared with the control. In the absence of a change in vesicle size or density, we conclude that the increased lipid content represents additional, newly formed vesicles.

We utilized recombinant DNA technology in order to elucidate those regions of cellugyrin that are important for vesicle biogenesis and the associated increase in synaptophysin targeting. Unexpectedly, we found that none of the hydrophilic regions of cellugyrin that are exposed to the cytoplasm are important for vesicle formation. This result suggests that cytoplasmic factors, such as adaptor proteins, may not be crucially important for the formation of microvesicles but may only be involved in cargo selection and recruitment to preformed vesicle buds. At the same time, two highly conserved short hydrophobic hairpins, one in the first luminal loop and another in the carboxyl terminus of cellugyrin, play key roles in this process. Another factor that is critical to cellugyrin function is the length (rather than the primary sequence) of the second luminal loop. We hypothesize that changing the length of this loop, similar to eliminating or scrambling of the short hydrophobic hairpins, alters the position of the vicinal transmembrane domains that may be crucial for protein function (see below).

At the same time, the primary sequence of the central cytoplasmic loop does not seem to be critical for cellugyrin function, whereas the length of that loop is a more flexible parameter than the length of the second luminal loop. One report on the C. elegans synaptogyrin homolog suggested that an arginine in the cytoplasmic loop was critical for targeting to nerve endings (24). An arginine is present in cellugyrin's cytoplasmic loop as well, but targeting of the protein to SLMVs was maintained despite scrambling the position of that amino acid (pHis-SCR104–112). It is possible therefore that targeting to nerve endings in C. elegans requires different molecular mechanisms than targeting to rat SLMVs.

The stringent requirements placed on the luminal loops in comparison with the cytoplasmic loop may suggest a role for cellugyrin in the induction of membrane curvature, which is believed to represent a crucial factor for vesicle biogenesis (25). Specifically, the luminal ends of the transmembrane domains may be required to be closer together than the cytosolic ends. According to one model, cellugyrin may assume an inverted cone structure (the base of the cone faces the cytosol) (Fig. 11) that may facilitate the initial budding process from the donor membrane. This model shares similarities with the proposed role of lysophosphatidic acid, an inverted cone-shaped lipid (8), which is thought to impart curvature to the nascent vesicle. On the contrary, synaptophysin's luminal loops are predicted to be 3 times as long as cellugyrin's luminal loops. Therefore, the synaptophysin molecule may not have a conical shape and hence does not induce membrane curvature and vesicle formation. The significance of the loop domains in determining the role played by four transmembrane domain proteins is supported by the fact that, whereas the sequences of the loops of all tetraspan vesicle proteins are highly variable, the lengths of these regions are highly conserved within each family (gyrins, physins, and SCAMPS) (26). Also, chimeras of cellugyrin and synaptophysin are unstable, whereas chimeras between cellugyrin and synaptogyrin, where the junction lies in this cytoplasmic loop, are stable and functional (data not shown).



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  		FIG. 11.
A model of cellugyrin's structure within the membrane. Asterisks, critical hydrophobic regions.

 
Mutagenesis experiments led to the identification of three stable cellugyrin mutants defective in SLMV targeting and facilitation of synaptophysin targeting (pMyc/His-CR1–177, pHis-SCR67–76, and pHis-MycinSCR142–151). It is striking that despite the different location of each mutation in the primary sequence, the observed defects in SLMV targeting and facilitation of synaptophysin targeting are relatively similar (i.e. all three constructs show a ~45% decrease in SLMV targeting with a ~75% decrease in synaptophysin facilitation). This pattern suggests that as the targeting capacity of cellugyrin is diminished by mutation, the recruitment of synaptophysin is more significantly impaired. Again, our model accounts for these results by suggesting that the mechanism for incorporating synaptophysin into the vesicle relies on efficient cellugyrin-dependent vesicle biogenesis. Interestingly, Pennuto et al. (27) recently reported that synaptophysin facilitates targeting of VAMP2 to synaptic vesicles in cultured hippocampal neurons. This finding may unveil the next step in the process of synaptic vesicle formation; synaptogyrin recruits synaptophysin, which, in turn, recruits VAMP2 to the vesicle. These studies may also shed light on a recent report that linked the inheritance of a nonsense mutation in the synaptogyrin 1 gene with schizophrenia (28). We may speculate that this patient cohort may be suffering due to a defect in synaptic vesicle biogenesis and/or cargo selection.

In conclusion, we have identified several functionally important regions in the cellugyrin molecule. We suggest that these regions are critical for the proper orientation of the transmembrane domains that, in turn, induce membrane curvature, which leads to the formation of new SLMVs in PC12 cells. Furthermore, based on our studies in COS7 cells, we suggest that although the role of cellugyrin in creating new vesicles may not depend on the cell type, further population of this compartment with individual proteins depends on cell-specific targeting mechanisms. Further studies will be geared toward the identification of the link between cellugyrin function and the cytoplasmic budding machinery.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01 DK52057 and R01 DK56736 (to K. V. K.) and by training grant T32 DK07201 (to G. M. B.). 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. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry, Boston University School of Medicine, K124D, 715 Albany St., Boston, MA 02118. Tel.: 617-638-5049; Fax: 617-638-5339; E-mail: kandror{at}biochem.bumc.bu.edu.

1 The abbreviations used are: SLMV, synaptic-like microvesicle; PNS, postnuclear supernatant(s); PAS, postabsorptive supernatant(s); TE, Triton elution; SE, SDS elution; TM, transmembrane; ER, endoplasmic reticulum. Back


   ACKNOWLEDGMENTS
 
We thank Dr. T. C. Südhof for the kind gift of the plasmid pCMV5-p38.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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