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Yeast homologues of tomosyn and lethal giant larvae function in exocytosis and are associated with the plasma membrane SNARE, Sec9.

Lehman K, Rossi G, Adamo JE, Brennwald P - J. Cell Biol. (1999)

Bottom Line: In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion.Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase.Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Weill Medical College of Cornell University, New York, New York 10021, USA.

ABSTRACT
We have identified a pair of related yeast proteins, Sro7p and Sro77p, based on their ability to bind to the plasma membrane SNARE (SNARE) protein, Sec9p. These proteins show significant similarity to the Drosophila tumor suppressor, lethal giant larvae and to the neuronal syntaxin-binding protein, tomosyn. SRO7 and SRO77 have redundant functions as loss of both gene products leads to a severe cold-sensitive growth defect that correlates with a severe defect in exocytosis. We show that similar to Sec9, Sro7/77 functions in the docking and fusion of post-Golgi vesicles with the plasma membrane. In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion. This demonstrates that the primary function of Sro7/77, and likely all members of the lethal giant larvae family, is in exocytosis rather than in regulating the actin cytoskeleton. Analysis of the association of Sro7p and Sec9p demonstrates that Sro7p directly interacts with Sec9p both in the cytosol and in the plasma membrane and can associate with Sec9p in the context of a SNAP receptor complex. Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase. Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.

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Identification of Sro7p and Sro77p as Sec9p-binding proteins. (A) Interaction of in vitro translated COOH-terminal domains of Sro7 and Sro77p with Sec9p. The COOH-terminal domain of Sro7p (left) identified in the two-hybrid screen and the homologous region of Sro77p were radiolabeled by coupled transcription/translation in rabbit reticulocyte lysates. In vitro translation reactions were incubated with various recombinant SNARE proteins immobilized on GST beads (each at ∼1 μM in the binding reaction), washed, and the bound material was analyzed by SDS-PAGE and autoradiography. The positions of molecular mass markers (expressed in kD) are indicated on the left of the gels. The bands corresponding to primary translation products of Sro7-CTp and Sro77-CTp are indicated by arrows. The lower molecular mass band presumably represents breakdown products. (B) Alignment of Sro7p and Sro77p with three lethal giant larvae family members and tomosyn: mouse LGL (MgCl-1, GenBank accession number D16141), human LGL (Hugl, GenBank accession number X86371), and Drosophila LGL (lg(2)l, Swiss protein accession number P08111) and tomosyn (accession number U92072). Each rectangle is drawn proportional to the length of each coding sequence. The positions of the predicted WD-40 repeats and the VAMP-like domain are indicated. The arrows indicate the region in Sro7p recovered by two-hybrid and used for the in vitro binding assays. The BLAST analysis shows the smallest sum probability (in parentheses) and highest scoring region (scores <10−3 are considered potentially significant).
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Figure 1: Identification of Sro7p and Sro77p as Sec9p-binding proteins. (A) Interaction of in vitro translated COOH-terminal domains of Sro7 and Sro77p with Sec9p. The COOH-terminal domain of Sro7p (left) identified in the two-hybrid screen and the homologous region of Sro77p were radiolabeled by coupled transcription/translation in rabbit reticulocyte lysates. In vitro translation reactions were incubated with various recombinant SNARE proteins immobilized on GST beads (each at ∼1 μM in the binding reaction), washed, and the bound material was analyzed by SDS-PAGE and autoradiography. The positions of molecular mass markers (expressed in kD) are indicated on the left of the gels. The bands corresponding to primary translation products of Sro7-CTp and Sro77-CTp are indicated by arrows. The lower molecular mass band presumably represents breakdown products. (B) Alignment of Sro7p and Sro77p with three lethal giant larvae family members and tomosyn: mouse LGL (MgCl-1, GenBank accession number D16141), human LGL (Hugl, GenBank accession number X86371), and Drosophila LGL (lg(2)l, Swiss protein accession number P08111) and tomosyn (accession number U92072). Each rectangle is drawn proportional to the length of each coding sequence. The positions of the predicted WD-40 repeats and the VAMP-like domain are indicated. The arrows indicate the region in Sro7p recovered by two-hybrid and used for the in vitro binding assays. The BLAST analysis shows the smallest sum probability (in parentheses) and highest scoring region (scores <10−3 are considered potentially significant).

Mentions: To corroborate the two-hybrid interaction between Sec9 and Sro7, we in vitro translated the region of Sro7 identified above in a rabbit reticulocyte lysate and examined its ability to interact with the immobilized GST-Sec9p fusion construct (see Materials and Methods for details about recombinant Sec9p construct). This COOH-terminal region of Sro7p specifically associated only with the GST-Sec9p construct (Fig. 1 A, left). Furthermore, Sro7p did not interact with GST alone, GST-Sso1p, or GST-Snc1p. Yeast contain a second gene, previously designated as SRO77, with 55% identity to SRO7 (Fig. 1 B), and has been shown to have overlapping function with SRO7 (Kagami et al. 1998). In a parallel experiment, we in vitro translated the corresponding domain of Sro77p and examined its ability to interact with GST fusion constructs. Sro77p associated with the GST construct containing Sec9p, but not significantly with GST-Sso1p and GST-Snc1p (Fig. 1 A, right).


Yeast homologues of tomosyn and lethal giant larvae function in exocytosis and are associated with the plasma membrane SNARE, Sec9.

Lehman K, Rossi G, Adamo JE, Brennwald P - J. Cell Biol. (1999)

Identification of Sro7p and Sro77p as Sec9p-binding proteins. (A) Interaction of in vitro translated COOH-terminal domains of Sro7 and Sro77p with Sec9p. The COOH-terminal domain of Sro7p (left) identified in the two-hybrid screen and the homologous region of Sro77p were radiolabeled by coupled transcription/translation in rabbit reticulocyte lysates. In vitro translation reactions were incubated with various recombinant SNARE proteins immobilized on GST beads (each at ∼1 μM in the binding reaction), washed, and the bound material was analyzed by SDS-PAGE and autoradiography. The positions of molecular mass markers (expressed in kD) are indicated on the left of the gels. The bands corresponding to primary translation products of Sro7-CTp and Sro77-CTp are indicated by arrows. The lower molecular mass band presumably represents breakdown products. (B) Alignment of Sro7p and Sro77p with three lethal giant larvae family members and tomosyn: mouse LGL (MgCl-1, GenBank accession number D16141), human LGL (Hugl, GenBank accession number X86371), and Drosophila LGL (lg(2)l, Swiss protein accession number P08111) and tomosyn (accession number U92072). Each rectangle is drawn proportional to the length of each coding sequence. The positions of the predicted WD-40 repeats and the VAMP-like domain are indicated. The arrows indicate the region in Sro7p recovered by two-hybrid and used for the in vitro binding assays. The BLAST analysis shows the smallest sum probability (in parentheses) and highest scoring region (scores <10−3 are considered potentially significant).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199738&req=5

Figure 1: Identification of Sro7p and Sro77p as Sec9p-binding proteins. (A) Interaction of in vitro translated COOH-terminal domains of Sro7 and Sro77p with Sec9p. The COOH-terminal domain of Sro7p (left) identified in the two-hybrid screen and the homologous region of Sro77p were radiolabeled by coupled transcription/translation in rabbit reticulocyte lysates. In vitro translation reactions were incubated with various recombinant SNARE proteins immobilized on GST beads (each at ∼1 μM in the binding reaction), washed, and the bound material was analyzed by SDS-PAGE and autoradiography. The positions of molecular mass markers (expressed in kD) are indicated on the left of the gels. The bands corresponding to primary translation products of Sro7-CTp and Sro77-CTp are indicated by arrows. The lower molecular mass band presumably represents breakdown products. (B) Alignment of Sro7p and Sro77p with three lethal giant larvae family members and tomosyn: mouse LGL (MgCl-1, GenBank accession number D16141), human LGL (Hugl, GenBank accession number X86371), and Drosophila LGL (lg(2)l, Swiss protein accession number P08111) and tomosyn (accession number U92072). Each rectangle is drawn proportional to the length of each coding sequence. The positions of the predicted WD-40 repeats and the VAMP-like domain are indicated. The arrows indicate the region in Sro7p recovered by two-hybrid and used for the in vitro binding assays. The BLAST analysis shows the smallest sum probability (in parentheses) and highest scoring region (scores <10−3 are considered potentially significant).
Mentions: To corroborate the two-hybrid interaction between Sec9 and Sro7, we in vitro translated the region of Sro7 identified above in a rabbit reticulocyte lysate and examined its ability to interact with the immobilized GST-Sec9p fusion construct (see Materials and Methods for details about recombinant Sec9p construct). This COOH-terminal region of Sro7p specifically associated only with the GST-Sec9p construct (Fig. 1 A, left). Furthermore, Sro7p did not interact with GST alone, GST-Sso1p, or GST-Snc1p. Yeast contain a second gene, previously designated as SRO77, with 55% identity to SRO7 (Fig. 1 B), and has been shown to have overlapping function with SRO7 (Kagami et al. 1998). In a parallel experiment, we in vitro translated the corresponding domain of Sro77p and examined its ability to interact with GST fusion constructs. Sro77p associated with the GST construct containing Sec9p, but not significantly with GST-Sso1p and GST-Snc1p (Fig. 1 A, right).

Bottom Line: In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion.Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase.Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Weill Medical College of Cornell University, New York, New York 10021, USA.

ABSTRACT
We have identified a pair of related yeast proteins, Sro7p and Sro77p, based on their ability to bind to the plasma membrane SNARE (SNARE) protein, Sec9p. These proteins show significant similarity to the Drosophila tumor suppressor, lethal giant larvae and to the neuronal syntaxin-binding protein, tomosyn. SRO7 and SRO77 have redundant functions as loss of both gene products leads to a severe cold-sensitive growth defect that correlates with a severe defect in exocytosis. We show that similar to Sec9, Sro7/77 functions in the docking and fusion of post-Golgi vesicles with the plasma membrane. In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion. This demonstrates that the primary function of Sro7/77, and likely all members of the lethal giant larvae family, is in exocytosis rather than in regulating the actin cytoskeleton. Analysis of the association of Sro7p and Sec9p demonstrates that Sro7p directly interacts with Sec9p both in the cytosol and in the plasma membrane and can associate with Sec9p in the context of a SNAP receptor complex. Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase. Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.

Show MeSH
Related in: MedlinePlus