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

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.

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).

Figure 2

SRO7 and SRO77 form a functionally redundant gene family and the sro7Δ, sro77Δ mutant exhibits a severe cold-sensitive phenotype. (A) Tetrad dissection of a cross between sro7Δ and sro77Δ single disruptants. Tetrads were dissected and grown on YPD plates at 25°C. Pinpoint colonies correspond to haploid segregants containing disruptions of both SRO7 and SRO77. Large colonies correspond to haploid segregants containing wild-type copies of both SRO7 and SRO77 or individual disruptions in either gene. T refers to a tetratype and NPD refers to nonparental ditype segregation patterns observed after scoring for auxotrophic markers for each disruption. (B) Cold sensitivity of the sro7Δ, sro77Δ double-disruptant strain. Each strain of a tetratype tetrad was struck out onto YPD plates and grown at either 37, 25, or 14°C. The genotype of the four strains is as indicated.

Figure 3

The sro7Δ, sro77Δ mutant has a pronounced defect in Golgi-to-cell surface transport. (A) Invertase secretion is deficient in the sro7Δ, sro77Δ mutant strain. Invertase secretion assays were performed after a temperature shift on several tetratype tetrads and a representative example is shown. The left frame shows a comparison of the percentage of invertase secreted into the periplasm after a 3-h shift to the restrictive temperature (19°C). The right frame shows the partitioning of invertase activity in the internal (intracellular) and external (periplasmic) fractions of these cells. All samples were measured in duplicate and the SD determined from two separate experiments. (B) The invertase that accumulates in sro7Δ, sro77Δ cells is fully glycosylated and transport of CPY to the vacuole is unaffected. Wild-type, single-disruptant, and double-disruptant strains, in addition to sec4-8 and sec18-1 strains, were grown and shifted as in A, except that sec4-8 and sec18-1 strains were grown for 2 h at 37°C before lysis. Equivalent amounts of each strain were lysed with glass beads, boiled in SDS sample buffer, subjected to SDS-PAGE, transferred to nitrocellulose, and probed with antibodies raised against invertase (top) or CPY (bottom). Mutants that block secretion after exit from the Golgi apparatus, such as sec4-8, accumulate internal pools of fully glycosylated invertase, whereas mutants that block at earlier stages, such as sec18-1, accumulate the core-glycosylated or ER-modified form of invertase. Likewise post-Golgi sec mutants do not affect CPY transport and maturation (and therefore show only the mature, mCPY form), unlike mutants that block earlier in the pathway such as sec18-1, which accumulates the core-glycosylated, p1 form of CPY. The forms of invertase and CPY found in the sro7Δ, sro77Δ cells suggest it is primarily affecting Golgi-to-cell surface transport.

Figure 4

(A) Loss of Sro7 and Sro77 leads to the accumulation of a large number of post-Golgi secretory vesicles at the nonpermissive temperature. The wild-type (SRO7, SRO77) and double-disruptant (sro7Δ, sro77Δ) strains were grown to mid-log phase at 37°C, shifted to the restrictive temperature of 19°C for 3 h, and then processed for thin section electron microscopy. High magnification micrographs of the accumulated vesicles demonstrated that >90% of the vesicles had diameters between 80 and 100 nm, identical to that reported for other post-Golgi sec mutants (Novick et al. 1980). (B) Actin localization in wild-type (SRO7, SRO77) and double-disruptant (sro7Δ, sro77Δ) strains. Haploid cells were grown overnight at the permissive temperature of 37°C to log phase, and then shifted to the restrictive temperature of 19°C for 3 h before fixing, permeabilizing, and staining with TRITC-phalloidin. Bars: A, 1 μm; B, 5 μm.

Figure 5

Sro7p is found in both cytosolic- and membrane-bound pools. (A) Affinity-purified antibodies to Sro7p recognize a protein of ∼105 kD on SDS-PAGE. Cells from wild-type (SRO7, SRO77), single disruptants (sro77Δ or sro7Δ), double disruptants (sro77Δ, sro7Δ), and strains containing high copy SRO7 (2μ SRO7) were spheroplasted, lysed, and boiled in SDS sample buffer. The samples were examined by 7% SDS-PAGE gels followed by immunoblotting with affinity-purified α-Sro7p antibodies. The higher molecular mass forms present in the SRO7 high copy lane probably represent denatured aggregates since they are much less apparent when samples are diluted before boiling and do not change appreciably during pulse-chase experiments (data not shown). (B) Sro7p is found in the soluble and membrane fractions of the cell. Cells containing vector only (pB23) or SRO7 on high copy (pB497) were grown to logarithmic phase, spheroplasted, lysed, and spun to remove unbroken cells. The lysate was treated with detergent or a mock control and subjected to two successive centrifugations: 30,000 g for 15 min, and then the S30 supernatant was centrifuged at 100,000 g for 1 h. Pellet fractions were resuspended in the same volumes as the supernatants to normalize. Samples from each fraction were boiled, analyzed by 7% SDS-PAGE, and immunoblotted with affinity-purified α-Sro7p antibody. Samples were also run on a 12.5% gel and immunoblotted with α-Sso1/2p polyclonal antibody as an internal control of the fractionation procedure.

Figure 6

Sro7p is associated with the plasma membrane in yeast. (A) Sro7p colocalizes with the plasma membrane marker Sso1/2p on a sucrose density gradient. Wild-type cells grown to logarithmic phase were spheroplasted, lysed, and spun at 30,000 g to pellet the membrane-associated pool of Sro7p that was analyzed on a 20–50% sucrose density gradient. The top shows the fractionation profile of Sro7p and the plasma membrane t-SNARE Sso1/2p in the gradient as determined by immunoblotting. The bottom shows the distribution of the GDPase activity (a Golgi marker) and cytochrome c reductase activity (an ER marker) in the gradient. Fraction 1 corresponds to the top of the gradient. Sro7p is found in two pools on the gradient: one that has been released from the membrane and is found at the top of gradient and a second pool that comigrates with the plasma membrane marker Sso1/2p. (B) Indirect immunofluorescence localization of Sro7p to the plasma membrane and soluble pool of the cell. Wild-type cells containing vector only (pB23) or SRO7 on high copy (pB497) were grown in selective media overnight and shifted in YPD for 2 h before fixing with formaldehyde. Cells were permeabilized using 0.05% SDS and stained with affinity-purified α-Sro7p antibody and detected using a rhodamine-conjugated α-rabbit secondary antibody. Stained cells were observed on a Zeiss LSM 510 confocal microscope and individual Z-slices were captured with LSM 510 software. Plasma membrane and cytosolic staining is evident in cells containing the high copy SRO7 but no specific staining was observed in the empty vector control cells containing a single copy of SRO7.

Figure 6

Sro7p is associated with the plasma membrane in yeast. (A) Sro7p colocalizes with the plasma membrane marker Sso1/2p on a sucrose density gradient. Wild-type cells grown to logarithmic phase were spheroplasted, lysed, and spun at 30,000 g to pellet the membrane-associated pool of Sro7p that was analyzed on a 20–50% sucrose density gradient. The top shows the fractionation profile of Sro7p and the plasma membrane t-SNARE Sso1/2p in the gradient as determined by immunoblotting. The bottom shows the distribution of the GDPase activity (a Golgi marker) and cytochrome c reductase activity (an ER marker) in the gradient. Fraction 1 corresponds to the top of the gradient. Sro7p is found in two pools on the gradient: one that has been released from the membrane and is found at the top of gradient and a second pool that comigrates with the plasma membrane marker Sso1/2p. (B) Indirect immunofluorescence localization of Sro7p to the plasma membrane and soluble pool of the cell. Wild-type cells containing vector only (pB23) or SRO7 on high copy (pB497) were grown in selective media overnight and shifted in YPD for 2 h before fixing with formaldehyde. Cells were permeabilized using 0.05% SDS and stained with affinity-purified α-Sro7p antibody and detected using a rhodamine-conjugated α-rabbit secondary antibody. Stained cells were observed on a Zeiss LSM 510 confocal microscope and individual Z-slices were captured with LSM 510 software. Plasma membrane and cytosolic staining is evident in cells containing the high copy SRO7 but no specific staining was observed in the empty vector control cells containing a single copy of SRO7.

Figure 7

Sro7p is not present on post-Golgi secretory vesicles. Cells from a BY29 (sec1-1, ura3-52) strain were shifted to 37°C in YP medium with 0.1% glucose for 2 h to accumulate post-Golgi vesicles and induce expression of invertase. Spheroplasts were prepared from these cells that were lysed osmotically by gentle resuspension in lysis buffer containing 0.8 M sorbitol. A 100,000 g membrane fraction (P3) enriched in post-Golgi vesicles was prepared from a 10,000 g supernatant fraction by differential centrifugation as described previously (Walworth and Novick 1987). The P3 vesicle fraction was resuspended in lysis buffer and layered onto a 20–40% sorbitol velocity gradient as described previously (Brennwald et al. 1994). The top shows the fractionation profile of latent invertase (a marker for secretory vesicles) compared with the distribution of total protein in the gradient. Invertase activity is expressed as micromoles of glucose per minute per fraction. Protein concentrations were determined according to Bradford 1976. The bottom shows the fractionation of Sro7p compared with Snc1/2, a marker for post-Golgi vesicles (Protopopov et al. 1993). The presence of Sro7p and Snc1/2 in the gradients was determined by immunoblotting followed by quantitation on a STORM PhosphorImager using ImageQuant software. Similar results were obtained using a P3 fraction prepared from a sec6-4 strain.

Figure 8

Full-length Sro7p, but not the COOH-terminal half of Sro7p, can be directly cross-linked to Sec9p in yeast lysates. Coprecipitation analysis was performed on radiolabeled yeast strains transformed with either myc-tagged YEpSEC9-myc plasmid (pB37) or, as a control, an otherwise identical untagged YEpSEC9 plasmid (pB37) in strains which contained a construct overexpressing either full-length Sro7 (pB363) or CT Sro7 (pB367) under control of the inducible GAL1 promoter. All strains were induced in galactose-containing media for 2 h before 1 h labeling of cells with ³⁵S-Express label in galactose-containing media. The cells were spheroplasted and lysed osmotically in PBS. Lysates were immediately subjected to treatment with the protein cleavable cross-linking agent (DSP) dissolved in DMSO or a DMSO control for 20 min on ice and subsequently boiled in 1% SDS buffer before dilution with IP buffer. Association because of cross-linking was monitored by a two-step immunoprecipitation protocol with the first immunoprecipitation being with the α-myc or directly with α-Sro7p antibodies (α-Sro7 IP). After washing, samples were boiled in 1% SDS/0.1 M DTT (to cleave the cross-linker), diluted with IP buffer, and subjected to a second round of immunoprecipitation with either α-Sro7p (top) or α-Sec9p (bottom) polyclonal antibodies. Samples were boiled in sample buffer, resolved by SDS-PAGE, dried, and exposed to film. The expression from the GAL1-SRO7 and GAL1-SRO7-CT constructs were similar (compare first and last lanes, marked α-Sro7 IP). A cross-linker and myc tag–dependent interaction is apparent between full-length Sro7p and Sec9p, strongly suggesting that these proteins are directly associated with each other in vivo. In contrast, the COOH-terminal domain does not show a detectable cross-linking in this assay.

Figure 9

Sro7p interacts with the post-Golgi SNARE proteins in detergent extracts. (A) Wild-type cells, grown to logarithmic phase were spheroplasted and lysed in buffer containing 0.5% NP-40. Detergent extracts were subjected to immunoprecipitations with either saturating amounts (3–10 μg of IgG/1 ml immunoprecipitation) of affinity-purified antibodies against either Snc1/2p, Sso1/2p, Sec9p, Sro7p, or an equivalent amount of IgG purified from a preimmune bleed of the respective rabbit. After recovery of immune complexes on protein A–Sepharose beads, beads were washed four times with the same buffer, and then boiled in SDS sample buffer. The samples were analyzed by SDS-PAGE, and then blotted with α-Sro7p, α-Sec9p, α-Snc1/2p, and α-Sso1/2p antibodies, respectively. To quantitate the samples that contained the same precipitating and immunoblotting antibody, we found it necessary to dilute these samples with SDS sample buffer before electrophoresis as follows: in the Snc1/2 blot, the Snc1/2 immunoprecipitates (and the control) were diluted 1:4; in the Sso1/2 blot, the Sso1/2 IPs were diluted 1:8; for the Sec9 blot, the Sec9 IPs were diluted 1:2; and for the Sro7 blot, the Sro7 IPs were diluted 1:2. A fraction of the total detergent lysate was immunoblotted in parallel to determine the total protein present in the immunoprecipitation reaction. The efficiency of the immunoprecipitation with saturating amounts of affinity-purified antibodies was calculated as follows: the amount of target protein immunoprecipitated by a given antibody was quantitated after immunoblotting and (after adjusting for dilution and amount loaded) divided by the amount of protein present in the total sample (after adjusting for the amount loaded). From this, the efficiency of precipitation by each antibody was determined to be as follows: α-Snc1/2p, 93%; α-Sso1/2p, 63%; α-Sec9p, 76%; and α-Sro7p, 98%. (B) Quantitation of the coimmunoprecipitations. The values are expressed as the amount of coimmunoprecipitating proteins present as a percentage of the total protein present (see above). ¹²⁵I–protein A signals on the immunoblots were quantitated on a Storm PhosphorImager using ImageQuant software. Note that the numbers that are expressed as a percentage of the total protein are higher for Sec9p than for the other three proteins. This is likely due to the fact that the levels of Sec9 protein found in wild-type yeast are 5–10-fold less than that of Snc1/2p and Sso1/2p, and at least threefold less than Sro7p. Thus, Sec9p is the limiting factor in these associations.

Figure 9

Sro7p interacts with the post-Golgi SNARE proteins in detergent extracts. (A) Wild-type cells, grown to logarithmic phase were spheroplasted and lysed in buffer containing 0.5% NP-40. Detergent extracts were subjected to immunoprecipitations with either saturating amounts (3–10 μg of IgG/1 ml immunoprecipitation) of affinity-purified antibodies against either Snc1/2p, Sso1/2p, Sec9p, Sro7p, or an equivalent amount of IgG purified from a preimmune bleed of the respective rabbit. After recovery of immune complexes on protein A–Sepharose beads, beads were washed four times with the same buffer, and then boiled in SDS sample buffer. The samples were analyzed by SDS-PAGE, and then blotted with α-Sro7p, α-Sec9p, α-Snc1/2p, and α-Sso1/2p antibodies, respectively. To quantitate the samples that contained the same precipitating and immunoblotting antibody, we found it necessary to dilute these samples with SDS sample buffer before electrophoresis as follows: in the Snc1/2 blot, the Snc1/2 immunoprecipitates (and the control) were diluted 1:4; in the Sso1/2 blot, the Sso1/2 IPs were diluted 1:8; for the Sec9 blot, the Sec9 IPs were diluted 1:2; and for the Sro7 blot, the Sro7 IPs were diluted 1:2. A fraction of the total detergent lysate was immunoblotted in parallel to determine the total protein present in the immunoprecipitation reaction. The efficiency of the immunoprecipitation with saturating amounts of affinity-purified antibodies was calculated as follows: the amount of target protein immunoprecipitated by a given antibody was quantitated after immunoblotting and (after adjusting for dilution and amount loaded) divided by the amount of protein present in the total sample (after adjusting for the amount loaded). From this, the efficiency of precipitation by each antibody was determined to be as follows: α-Snc1/2p, 93%; α-Sso1/2p, 63%; α-Sec9p, 76%; and α-Sro7p, 98%. (B) Quantitation of the coimmunoprecipitations. The values are expressed as the amount of coimmunoprecipitating proteins present as a percentage of the total protein present (see above). ¹²⁵I–protein A signals on the immunoblots were quantitated on a Storm PhosphorImager using ImageQuant software. Note that the numbers that are expressed as a percentage of the total protein are higher for Sec9p than for the other three proteins. This is likely due to the fact that the levels of Sec9 protein found in wild-type yeast are 5–10-fold less than that of Snc1/2p and Sso1/2p, and at least threefold less than Sro7p. Thus, Sec9p is the limiting factor in these associations.

Figure 10

Sro7p interacts specifically with Sec9p in the cytoplasm and at the plasma membrane. (A) Cells containing high copy SEC9 (pB35) were grown to logarithmic phase, spheroplasted, and lysed. The lysate was centrifuged at 30,000 g to separate cytosol and membrane fractions, the samples were adjusted to 0.5% NP-40, centrifuged at 13,000 g for 10 min to eliminate insoluble material, and subjected to coimmunoprecipitation analysis as in Fig. 8. Samples were analyzed by SDS-PAGE and immunoblotted with α-Sec9p, α-Sro7p, α-Sso1/2p, and α-Snc1/2p antibodies. (B) Serial coprecipitation analysis demonstrates that Sro7p interacts with Sso1/2p and Snc1/2p only through the interaction with Sec9p. A 30,000 g membrane fraction was solubilized with detergent as above and was precleared of Sec9 protein by immunoprecipitation with saturating amounts of affinity-purified α-Sec9p antibody or equivalent amounts of preimmune IgG as a control. Fractions were subjected to a second round of immunoprecipitations with α-Sec9p, α-Sro7p, α-Sso1/2p, and α-Snc1/2p polyclonal antibodies and analyzed as above.

Figure 11

Suppression of the rho3 deletion by multicopy SRO7 and SEC9. A diploid heterozygous for a disruption of the chromosomal rho3 gene (a/α, rho3Δ::LEU2/RHO3, leu2-3/leu2-3; ura3-52/ura3-52) was transformed with multicopy plasmids containing (top) empty vector (pB23), (middle) SRO7 (pB427), or (bottom) SEC9 (pB35). Tetrads were dissected on YPD plates and grown for 3 d at 25°C.