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Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells.

Tucker WC, Edwardson JM, Bai J, Kim HJ, Martin TF, Chapman ER - J. Cell Biol. (2003)

Bottom Line: Several putative Ca2+-syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown.Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I-XI to interfere with endogenous syt-effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells.As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Wisconsin, Madison, WI 53706, USA.

ABSTRACT
The synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca2+-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca2+-syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I-XI to interfere with endogenous syt-effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin-SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP2)-binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca2+-regulated interactions with t-SNAREs and PIP2, target molecules known to play critical roles in exocytosis.

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Inhibition of secretion by C2A domains is correlated with PIP2-binding activity. GST fusions of the indicated C2A isoforms (0.13 nmole) were immobilized on glutathione-Sepharose beads and assayed for 3H-labeled liposome binding in the presence of 2 mM EGTA (white bars) or 100 μM Ca2+ (black bars) as described in Materials and methods. (A) PIP2-binding properties of C2A from syt I–XI. Liposomes were composed of 1.5% PIP2/98.5% PC. (B) The extent of inhibition (Fig. 1 B) was plotted versus the extent of PIP2-binding activity at 100 μM Ca2+ (A). Data were fitted by linear regression; r2 values and P values are reported. (C) PS-binding properties of C2A from syt I–XI. Liposomes were composed of 25% PS/ 75% PC. (D) The extent of inhibition (Fig. 1 B) was plotted versus the extent of PS-binding activity at 100 μM Ca2+ (C). Note that C2A-I and -II exhibit robust PS-binding activity but fail to inhibit release. (E) Kinetic studies of C2A–PS/PC liposome interactions. Fluorescence resonance energy transfer between native tryptophan residue(s) (donor) in C2A and dansyl-PE incorporated into liposomes (acceptor; 5% with 25% PS/70% PC) was used to monitor the time course of C2A–PS/PC membrane interactions as described previously (Davis et al., 1999). Kinetic traces for each C2A (3 μM [final]) were obtained by rapid mixing with liposomes (11 nM [final]) plus Ca2+ (100 μM [final]). The observed rates (kobs) were determined by fitting the data with a single exponential function for C2A-I and -III; data for C2A-VII were best fitted by a double exponential function. (Inset) kobs is plotted as a function of the indicated liposome concentration. Note the kobs of the slower component (○) of C2A-VII is not dependent on liposome concentration, indicating that it represents a conformational change or complex rearrangement, such as oligomerization (Fukuda and Mikoshiba, 2001; Wu et al., 2003), after C2A–liposome complex assembly; only the fast component (•) is considered here. Error bars represent that SD from three independent experiments. (F) PIP2 enhances the affinity of C2A-III and C2A-VII, but not C2A-I, for membranes. Values for koff, kon, and KD of the C2A–liposome interaction were calculated as described previously (Davis et al., 1999). C2A-I, -III, and -VII bind to liposomes containing 25% PS (with 70% PC and 5% dansyl-PE) with similar rate constants and affinities, whereas C2A-III and -VII exhibit a three- to fourfold lower KD for liposomes containing 1.5% PIP2 and 25% PS (with 68.5% PC and 5% dansyl-PE).
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fig2: Inhibition of secretion by C2A domains is correlated with PIP2-binding activity. GST fusions of the indicated C2A isoforms (0.13 nmole) were immobilized on glutathione-Sepharose beads and assayed for 3H-labeled liposome binding in the presence of 2 mM EGTA (white bars) or 100 μM Ca2+ (black bars) as described in Materials and methods. (A) PIP2-binding properties of C2A from syt I–XI. Liposomes were composed of 1.5% PIP2/98.5% PC. (B) The extent of inhibition (Fig. 1 B) was plotted versus the extent of PIP2-binding activity at 100 μM Ca2+ (A). Data were fitted by linear regression; r2 values and P values are reported. (C) PS-binding properties of C2A from syt I–XI. Liposomes were composed of 25% PS/ 75% PC. (D) The extent of inhibition (Fig. 1 B) was plotted versus the extent of PS-binding activity at 100 μM Ca2+ (C). Note that C2A-I and -II exhibit robust PS-binding activity but fail to inhibit release. (E) Kinetic studies of C2A–PS/PC liposome interactions. Fluorescence resonance energy transfer between native tryptophan residue(s) (donor) in C2A and dansyl-PE incorporated into liposomes (acceptor; 5% with 25% PS/70% PC) was used to monitor the time course of C2A–PS/PC membrane interactions as described previously (Davis et al., 1999). Kinetic traces for each C2A (3 μM [final]) were obtained by rapid mixing with liposomes (11 nM [final]) plus Ca2+ (100 μM [final]). The observed rates (kobs) were determined by fitting the data with a single exponential function for C2A-I and -III; data for C2A-VII were best fitted by a double exponential function. (Inset) kobs is plotted as a function of the indicated liposome concentration. Note the kobs of the slower component (○) of C2A-VII is not dependent on liposome concentration, indicating that it represents a conformational change or complex rearrangement, such as oligomerization (Fukuda and Mikoshiba, 2001; Wu et al., 2003), after C2A–liposome complex assembly; only the fast component (•) is considered here. Error bars represent that SD from three independent experiments. (F) PIP2 enhances the affinity of C2A-III and C2A-VII, but not C2A-I, for membranes. Values for koff, kon, and KD of the C2A–liposome interaction were calculated as described previously (Davis et al., 1999). C2A-I, -III, and -VII bind to liposomes containing 25% PS (with 70% PC and 5% dansyl-PE) with similar rate constants and affinities, whereas C2A-III and -VII exhibit a three- to fourfold lower KD for liposomes containing 1.5% PIP2 and 25% PS (with 68.5% PC and 5% dansyl-PE).

Mentions: Ca2+ triggers the penetration of the Ca2+-binding loops of syt into the surface of lipid bilayers that contain anionic phospholipids (e.g., phosphatidylserine [PS]), and this interaction has been proposed to function as a coupling step in excitation-secretion coupling (Bai et al., 2002). Whereas PS-binding activity has been characterized for the isolated C2A domains of syts I–VIII (Li et al., 1995; Sugita et al., 2002), little is known concerning the interaction of syts with other lipids, particularly PIP2, which has been shown to bind syt I (Schiavo et al., 1996). PIP2 is a plasma membrane lipid that is essential for exocytosis of LDCVs (Eberhard et al., 1990; Hay and Martin, 1993; Hay et al., 1995), potentially via its interactions with syt. Therefore, we screened the C2A domains used in the inhibition studies for PS- and PIP2-binding activity (Fig. 2, A and C). With the exception of isoforms IV, VIII, and XI, the C2A domains of the syt family bound PS-containing liposomes in response to Ca2+ (Fig. 2 C). The PIP2-binding profile, however, was dramatically different (Fig. 2 A). PIP2-containing liposomes bound C2A-III and C2A-VII and to a lesser extent C2A-V and C2A-X. Hence, PIP2–C2A interactions are restricted to fewer syt isoforms than is PS-binding activity. However, as detailed below, in some syt isoforms PIP2 binding is mediated by the C2B domain (see Fig. 6; Schiavo et al., 1996).


Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells.

Tucker WC, Edwardson JM, Bai J, Kim HJ, Martin TF, Chapman ER - J. Cell Biol. (2003)

Inhibition of secretion by C2A domains is correlated with PIP2-binding activity. GST fusions of the indicated C2A isoforms (0.13 nmole) were immobilized on glutathione-Sepharose beads and assayed for 3H-labeled liposome binding in the presence of 2 mM EGTA (white bars) or 100 μM Ca2+ (black bars) as described in Materials and methods. (A) PIP2-binding properties of C2A from syt I–XI. Liposomes were composed of 1.5% PIP2/98.5% PC. (B) The extent of inhibition (Fig. 1 B) was plotted versus the extent of PIP2-binding activity at 100 μM Ca2+ (A). Data were fitted by linear regression; r2 values and P values are reported. (C) PS-binding properties of C2A from syt I–XI. Liposomes were composed of 25% PS/ 75% PC. (D) The extent of inhibition (Fig. 1 B) was plotted versus the extent of PS-binding activity at 100 μM Ca2+ (C). Note that C2A-I and -II exhibit robust PS-binding activity but fail to inhibit release. (E) Kinetic studies of C2A–PS/PC liposome interactions. Fluorescence resonance energy transfer between native tryptophan residue(s) (donor) in C2A and dansyl-PE incorporated into liposomes (acceptor; 5% with 25% PS/70% PC) was used to monitor the time course of C2A–PS/PC membrane interactions as described previously (Davis et al., 1999). Kinetic traces for each C2A (3 μM [final]) were obtained by rapid mixing with liposomes (11 nM [final]) plus Ca2+ (100 μM [final]). The observed rates (kobs) were determined by fitting the data with a single exponential function for C2A-I and -III; data for C2A-VII were best fitted by a double exponential function. (Inset) kobs is plotted as a function of the indicated liposome concentration. Note the kobs of the slower component (○) of C2A-VII is not dependent on liposome concentration, indicating that it represents a conformational change or complex rearrangement, such as oligomerization (Fukuda and Mikoshiba, 2001; Wu et al., 2003), after C2A–liposome complex assembly; only the fast component (•) is considered here. Error bars represent that SD from three independent experiments. (F) PIP2 enhances the affinity of C2A-III and C2A-VII, but not C2A-I, for membranes. Values for koff, kon, and KD of the C2A–liposome interaction were calculated as described previously (Davis et al., 1999). C2A-I, -III, and -VII bind to liposomes containing 25% PS (with 70% PC and 5% dansyl-PE) with similar rate constants and affinities, whereas C2A-III and -VII exhibit a three- to fourfold lower KD for liposomes containing 1.5% PIP2 and 25% PS (with 68.5% PC and 5% dansyl-PE).
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Related In: Results  -  Collection

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fig2: Inhibition of secretion by C2A domains is correlated with PIP2-binding activity. GST fusions of the indicated C2A isoforms (0.13 nmole) were immobilized on glutathione-Sepharose beads and assayed for 3H-labeled liposome binding in the presence of 2 mM EGTA (white bars) or 100 μM Ca2+ (black bars) as described in Materials and methods. (A) PIP2-binding properties of C2A from syt I–XI. Liposomes were composed of 1.5% PIP2/98.5% PC. (B) The extent of inhibition (Fig. 1 B) was plotted versus the extent of PIP2-binding activity at 100 μM Ca2+ (A). Data were fitted by linear regression; r2 values and P values are reported. (C) PS-binding properties of C2A from syt I–XI. Liposomes were composed of 25% PS/ 75% PC. (D) The extent of inhibition (Fig. 1 B) was plotted versus the extent of PS-binding activity at 100 μM Ca2+ (C). Note that C2A-I and -II exhibit robust PS-binding activity but fail to inhibit release. (E) Kinetic studies of C2A–PS/PC liposome interactions. Fluorescence resonance energy transfer between native tryptophan residue(s) (donor) in C2A and dansyl-PE incorporated into liposomes (acceptor; 5% with 25% PS/70% PC) was used to monitor the time course of C2A–PS/PC membrane interactions as described previously (Davis et al., 1999). Kinetic traces for each C2A (3 μM [final]) were obtained by rapid mixing with liposomes (11 nM [final]) plus Ca2+ (100 μM [final]). The observed rates (kobs) were determined by fitting the data with a single exponential function for C2A-I and -III; data for C2A-VII were best fitted by a double exponential function. (Inset) kobs is plotted as a function of the indicated liposome concentration. Note the kobs of the slower component (○) of C2A-VII is not dependent on liposome concentration, indicating that it represents a conformational change or complex rearrangement, such as oligomerization (Fukuda and Mikoshiba, 2001; Wu et al., 2003), after C2A–liposome complex assembly; only the fast component (•) is considered here. Error bars represent that SD from three independent experiments. (F) PIP2 enhances the affinity of C2A-III and C2A-VII, but not C2A-I, for membranes. Values for koff, kon, and KD of the C2A–liposome interaction were calculated as described previously (Davis et al., 1999). C2A-I, -III, and -VII bind to liposomes containing 25% PS (with 70% PC and 5% dansyl-PE) with similar rate constants and affinities, whereas C2A-III and -VII exhibit a three- to fourfold lower KD for liposomes containing 1.5% PIP2 and 25% PS (with 68.5% PC and 5% dansyl-PE).
Mentions: Ca2+ triggers the penetration of the Ca2+-binding loops of syt into the surface of lipid bilayers that contain anionic phospholipids (e.g., phosphatidylserine [PS]), and this interaction has been proposed to function as a coupling step in excitation-secretion coupling (Bai et al., 2002). Whereas PS-binding activity has been characterized for the isolated C2A domains of syts I–VIII (Li et al., 1995; Sugita et al., 2002), little is known concerning the interaction of syts with other lipids, particularly PIP2, which has been shown to bind syt I (Schiavo et al., 1996). PIP2 is a plasma membrane lipid that is essential for exocytosis of LDCVs (Eberhard et al., 1990; Hay and Martin, 1993; Hay et al., 1995), potentially via its interactions with syt. Therefore, we screened the C2A domains used in the inhibition studies for PS- and PIP2-binding activity (Fig. 2, A and C). With the exception of isoforms IV, VIII, and XI, the C2A domains of the syt family bound PS-containing liposomes in response to Ca2+ (Fig. 2 C). The PIP2-binding profile, however, was dramatically different (Fig. 2 A). PIP2-containing liposomes bound C2A-III and C2A-VII and to a lesser extent C2A-V and C2A-X. Hence, PIP2–C2A interactions are restricted to fewer syt isoforms than is PS-binding activity. However, as detailed below, in some syt isoforms PIP2 binding is mediated by the C2B domain (see Fig. 6; Schiavo et al., 1996).

Bottom Line: Several putative Ca2+-syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown.Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I-XI to interfere with endogenous syt-effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells.As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Wisconsin, Madison, WI 53706, USA.

ABSTRACT
The synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca2+-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca2+-syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I-XI to interfere with endogenous syt-effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin-SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP2)-binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca2+-regulated interactions with t-SNAREs and PIP2, target molecules known to play critical roles in exocytosis.

Show MeSH
Related in: MedlinePlus