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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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The C2B and C2A-C2B domains from syt I and syt IX inhibit exocytosis. C2A, C2B, and C2A-C2B domains (10 μM) of syt isoforms I and IX were assayed for their ability to inhibit exocytosis as described in Fig. 1 B.
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fig5: The C2B and C2A-C2B domains from syt I and syt IX inhibit exocytosis. C2A, C2B, and C2A-C2B domains (10 μM) of syt isoforms I and IX were assayed for their ability to inhibit exocytosis as described in Fig. 1 B.

Mentions: Recombinant C2B-I copurifies with a bacterial contaminant that appears to be RNA (Ubach et al., 2001; Wu et al., 2003; unpublished data). Others have reported that removal of this contaminant disrupts inhibition mediated by C2B-I (Sugita et al., 2001; Shin et al., 2003). For our studies, recombinant proteins including t-SNAREs and C2 domains were treated with nucleases and high salt buffers, which completely remove bacterial contaminants. We found that contaminant-free C2B-I retained inhibitory activity (Fig. 5; Desai et al., 2000). Thus, inhibition of secretion is not an artifact due to the presence of the contaminant.


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)

The C2B and C2A-C2B domains from syt I and syt IX inhibit exocytosis. C2A, C2B, and C2A-C2B domains (10 μM) of syt isoforms I and IX were assayed for their ability to inhibit exocytosis as described in Fig. 1 B.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172790&req=5

fig5: The C2B and C2A-C2B domains from syt I and syt IX inhibit exocytosis. C2A, C2B, and C2A-C2B domains (10 μM) of syt isoforms I and IX were assayed for their ability to inhibit exocytosis as described in Fig. 1 B.
Mentions: Recombinant C2B-I copurifies with a bacterial contaminant that appears to be RNA (Ubach et al., 2001; Wu et al., 2003; unpublished data). Others have reported that removal of this contaminant disrupts inhibition mediated by C2B-I (Sugita et al., 2001; Shin et al., 2003). For our studies, recombinant proteins including t-SNAREs and C2 domains were treated with nucleases and high salt buffers, which completely remove bacterial contaminants. We found that contaminant-free C2B-I retained inhibitory activity (Fig. 5; Desai et al., 2000). Thus, inhibition of secretion is not an artifact due to the presence of the contaminant.

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