FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2122724972> ?p ?o ?g. }

• W2122724972 endingPage "36971" @default.
• W2122724972 startingPage "36962" @default.
• W2122724972 abstract "Both the delivery of secretory vesicles and asymmetric distribution of mRNA to the bud are dependent upon the actin cytoskeleton in yeast. Here we examined whether components of the exocytic apparatus play a role in mRNA transport. By screening secretion mutants in situ and in vivo, we found that all had an altered pattern of ASH1 mRNA localization. These included alleles of CDC42 and RHO3 (cdc42-6 and rho3-V51) thought to regulate specifically the fusion of secretory vesicles but were found to affect strongly the cytoskeleton as well. Most interestingly, mutations in late secretion-related genes not directly involved in actin regulation also showed substantial alterations in ASH1 mRNA distribution. These included mutations in genes encoding components of the exocyst (SEC10 and SEC15), SNARE regulatory proteins (SEC1, SEC4, and SRO7), SNAREs (SEC9 and SSO1/2), and proteins involved in Golgi export (PIK1 and YPT31/32). Importantly, prominent defects in the actin cytoskeleton were observed in all of these strains, thus implicating a known causal relationship between the deregulation of actin and the inhibition of mRNA transport. Our novel observations suggest that vesicular transport regulates the actin cytoskeleton in yeast (and not just vice versa) leading to subsequent defects in mRNA transport and localization. Both the delivery of secretory vesicles and asymmetric distribution of mRNA to the bud are dependent upon the actin cytoskeleton in yeast. Here we examined whether components of the exocytic apparatus play a role in mRNA transport. By screening secretion mutants in situ and in vivo, we found that all had an altered pattern of ASH1 mRNA localization. These included alleles of CDC42 and RHO3 (cdc42-6 and rho3-V51) thought to regulate specifically the fusion of secretory vesicles but were found to affect strongly the cytoskeleton as well. Most interestingly, mutations in late secretion-related genes not directly involved in actin regulation also showed substantial alterations in ASH1 mRNA distribution. These included mutations in genes encoding components of the exocyst (SEC10 and SEC15), SNARE regulatory proteins (SEC1, SEC4, and SRO7), SNAREs (SEC9 and SSO1/2), and proteins involved in Golgi export (PIK1 and YPT31/32). Importantly, prominent defects in the actin cytoskeleton were observed in all of these strains, thus implicating a known causal relationship between the deregulation of actin and the inhibition of mRNA transport. Our novel observations suggest that vesicular transport regulates the actin cytoskeleton in yeast (and not just vice versa) leading to subsequent defects in mRNA transport and localization. The establishment of cell polarity in eukaryotes involves the asymmetric organization of mRNA, the cytoskeleton, and the secretory pathway to lead to the polarized distribution of new membrane along a given axis (1Drubin D.G. Nelson W.J. Cell. 1996; 84: 335-344Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 2Kloc M. Zearfoss N.R. Etkin L.D. Cell. 2002; 108: 533-544Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). In yeast, polarization leads to the budding of daughter cells (cell division), the asymmetric segregation of cell-fate determinants, and mating of haplotypes (3Casamayor A. Snyder M. Curr. Opin. Microbiol. 2002; 2: 179-186Crossref Scopus (127) Google Scholar, 4Bretscher A. J. Cell Biol. 2003; 160: 811-816Crossref PubMed Scopus (119) Google Scholar). These aspects of polarization require proper control of the actin cytoskeleton, as mutations therein block the exocytosis of proteins and new membrane along the axis of growth as well as the delivery of an mRNA encoding a protein involved in mating-type control (e.g. Ash1). For example, loss-of-function mutations in genes encoding yeast actin (ACT1), tropomyosin (TPM1,2), and a type V myosin (MYO2) all block exocytosis and result in lethality (5Novick P. Botstein D. Cell. 1985; 40: 405-416Abstract Full Text PDF PubMed Scopus (365) Google Scholar, 6Pruyne D.W. Schott D.H. Bretscher A. J. Cell Biol. 1998; 143: 1931-1945Crossref PubMed Scopus (277) Google Scholar, 7Johnston G.C. Prendergast J.A. Singer R.A. J. Cell Biol. 1991; 113: 539-551Crossref PubMed Scopus (386) Google Scholar). Similarly, mutations in actin, tropomyosin, and another type V myosin (encoded by MYO4) also block ASH1 mRNA transport (2Kloc M. Zearfoss N.R. Etkin L.D. Cell. 2002; 108: 533-544Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 8Long R.M. Singer R.H. Meng X. Gonzalez I. Nasmyth K. Jansen R.P. Science. 1997; 277: 383-387Crossref PubMed Scopus (416) Google Scholar, 9Takizawa P.A. Sil A. Swedlow J.R. Herskowitz I. Vale R.D. Nature. 1997; 389: 90-93Crossref PubMed Scopus (313) Google Scholar, 10Jansen R.P. FASEB J. 1999; 13: 455-466Crossref PubMed Scopus (139) Google Scholar, 11Chartrand P. Singer R.H. Long R.M. Annu. Rev. Cell Dev. Biol. 2001; 17: 297-310Crossref PubMed Scopus (62) Google Scholar). Thus, an essentially common mechanism for both vesicle and mRNA transport to the growing bud appears to have evolved in eukaryotes. A key question that remains to be resolved is whether components of the secretory apparatus play a role in mRNA transport and localization to the bud. Because both share an actin-dependent transport mechanism, it seems likely that feedback from the exocytic pathway may affect actin assembly and, therefore, subsequent mRNA transport. In yeast, SHE4, one of five SHE genes known to be required for the transport and localization of ASH1 mRNA (2Kloc M. Zearfoss N.R. Etkin L.D. Cell. 2002; 108: 533-544Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 9Takizawa P.A. Sil A. Swedlow J.R. Herskowitz I. Vale R.D. Nature. 1997; 389: 90-93Crossref PubMed Scopus (313) Google Scholar, 10Jansen R.P. FASEB J. 1999; 13: 455-466Crossref PubMed Scopus (139) Google Scholar, 11Chartrand P. Singer R.H. Long R.M. Annu. Rev. Cell Dev. Biol. 2001; 17: 297-310Crossref PubMed Scopus (62) Google Scholar, 12Jansen R.P. Dowzer C. Michaelis C. Galova M. Nasmyth K. Cell. 1996; 84: 687-697Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), was isolated as an endocytosis-defective mutant and was shown to have a depolarized actin cytoskeleton (13Wendland B. McCaffery J.M. Xiao Q. Emr S. J. Cell Biol. 1996; 135: 1485-1500Crossref PubMed Scopus (202) Google Scholar). Thus, components of the endocytic apparatus that regulate actin may also influence mRNA transport. Until recently, the function of She4 in either endocytosis or actin regulation was not known. However, it was shown that She4 interacts directly with the motor domain of unconventional myosins, including Myo4/She1, and that mutations in this domain can bypass she4Δ growth and endocytosis defects but not defects in mating-type switching (14Toi H. Fujimura-Kamada K. Irie K. Takai Y. Todo S. Tanaka K. Mol. Biol. Cell. 2003; 14: 2237-2249Crossref PubMed Scopus (58) Google Scholar). This suggests that She4 may serve multiple roles, one in the regulation of myosin motor function and perhaps an additional role in ASH1 mRNA transport (14Toi H. Fujimura-Kamada K. Irie K. Takai Y. Todo S. Tanaka K. Mol. Biol. Cell. 2003; 14: 2237-2249Crossref PubMed Scopus (58) Google Scholar). These are likely to be mediated by different domains of the protein, as expression of the C-terminal domain of She4 suppresses the growth defects in the she4Δ mutant, whereas full-length She4 is necessary to suppress both the growth and the HO gene expression phenotypes (14Toi H. Fujimura-Kamada K. Irie K. Takai Y. Todo S. Tanaka K. Mol. Biol. Cell. 2003; 14: 2237-2249Crossref PubMed Scopus (58) Google Scholar). Other endocytic proteins have been shown to modulate mRNA transport and localization, including Drosophila Rab11 which plays a role in endocytic protein sorting, microtubule organization, and also oskar mRNA localization (15Dollar G. Struckhoff E. Michaud J. Cohen R. Development (Camb.). 2002; 129: 517-526PubMed Google Scholar). Thus, a connection between endocytic transport, regulation of the microtubule cytoskeleton, and mRNA localization exists in higher organisms and may parallel that observed in yeast. Here we have examined directly whether mutations in components of the late secretory pathway affect the transport of ASH1 mRNA to the daughter cell and its localization therein using both in situ and in vivo assays. Most importantly, we have found that both polarity and secretion-related proteins modulate the asymmetric distribution of ASH1 mRNA. For example, alleles of small GTPases (e.g. cdc42-6 and rho3-V51) thought to mediate specifically secretory vesicle fusion and not to affect significantly the actin cytoskeleton (16Adamo J.E. Rossi G. Brennwald P. Mol. Biol. Cell. 1999; 12: 4121-4133Crossref Scopus (174) Google Scholar, 17Adamo J.E. Moskow J.J. Gladfelter A.S. Viterbo D. Lew D.J. Brennwald P.J. J. Cell Biol. 2001; 155: 581-592Crossref PubMed Scopus (137) Google Scholar) greatly inhibited ASH1 distribution to the bud. This effect was also observed in yeast bearing mutations in either RAS2 (e.g. ras2Δ) or CMD1 (e.g. cmd1-239), although we note that the actin cytoskeleton was substantially disorganized in all of these mutants. Most surprisingly, however, conditional loss-of-function mutations in genes thought to be directly involved in protein export, and not actin regulation, revealed numerous cases whereby mRNA localization was also affected. For example, mutations in genes encoding proteins involved in the transport and fusion of secretory vesicles, like Sec4 and Sro7 (18Salminen A. Novick P.J. Cell. 1987; 49: 527-538Abstract Full Text PDF PubMed Scopus (593) Google Scholar, 19Lehman K. Rossi G. Adamo J.E. Brennwald P. J. Cell Biol. 1999; 146: 125-140Crossref PubMed Scopus (175) Google Scholar) as well as Pik1, a phosphatidylinositol 4-kinase involved in Golgi export (20Flanagan C.A. Schnieders E.A. Emerick A.W. Kunisawa R. Admon A. Thorner J. Science. 1993; 262: 1444-1448Crossref PubMed Scopus (171) Google Scholar, 21Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 8: 2673-2689Crossref Scopus (286) Google Scholar), all greatly inhibited ASH1 mRNA distribution to the bud. To a somewhat lesser extent, mutations in the Sec1 SNARE 1The abbreviations used are: SNARE, soluble NSF attachment protein receptor; GFP, green fluorescent protein; PBS, phosphate-buffered saline; FISH, fluorescence in situ hybridization; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. regulator (22Jahn R. Neuron. 2000; 2: 201-204Abstract Full Text Full Text PDF Scopus (91) Google Scholar), the exocyst components, Sec10 and Sec15 (23TerBush D.R. Maurice T. Roth D. Novick P. EMBO J. 1996; 15: 6483-6494Crossref PubMed Scopus (680) Google Scholar) (the latter being a possible Sec4 effector (24Salminen A. Novick P.J. J. Cell Biol. 1989; 109: 1023-1036Crossref PubMed Scopus (99) Google Scholar)), and the exocytic SNAREs, Sec9, and Sso1/2 (25Brennwald P. Kearns B. Champion K. Keranen S. Bankaitis V. Novick P. Cell. 1994; 79: 245-258Abstract Full Text PDF PubMed Scopus (311) Google Scholar, 26Aalto M.K. Ronne H. Keranen S. EMBO J. 1993; 11: 4095-4104Crossref Scopus (344) Google Scholar), also resulted in the mislocalization of ASH1 mRNA. This suggests that SNAREs, SNARE regulatory, exocyst, and Golgi export proteins all exert control over mRNA transport and localization. Subsequent analysis of the actin cytoskeleton in these mutants revealed that all have a disorganized pattern of actin labeling, which occurs rapidly upon the shift to semi-restrictive and restrictive temperatures. This indicates that a novel relationship exists between vesicle transport and the integrity of the actin cytoskeleton. Thus, active exocytosis in yeast is necessary for both maintenance of the polarized actin cytoskeleton and subsequent mRNA transport and localization to the growing bud. Media, DNA, and Genetic Manipulations—Yeast were grown in standard growth media containing 2% glucose. Synthetic complete and drop-out media were prepared similar to that described previously (27Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). Standard methods were used for the introduction of DNA into yeast and the preparation of genomic DNA (27Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). Cells transformed with plasmids for ASH1 mRNA labeling (see below) were grown on selective synthetic medium. For the induction of MS2 coat protein fused to GFP (MS2-GFP), cells were switched to synthetic medium lacking methionine for 1-2 h. Yeast Strains—Yeast strains used are listed in Table I. Plasmids—Plasmids encoding an ASH1 gene fragment with an MS2 viral coat protein-binding site in its 3′-untranslated region (pIIIA/ASH1-UTR) and a gene fusion of the MS2 coat protein and GFP (pCPGFP) (28Beach D.L. Salmon E.D. Bloom K. Curr. Biol. 1999; 9: 569-578Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) were generously provided by Kerry Bloom (University of North Carolina, Chapel Hill).Table IYeast strainsStrainGenotypeSourceW303-1aMATaade2 can1 his3 leu2 lys2 trp1 ura3J. HirschBY4741MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0EuroscarfSEY6210MATα his3-Δ 200 leu2-3, Δ112 lys2 Δ801 trp1-901 suc2-Δ9 arf1Δ::HIS3S. EmrCdc24-1MATaleu2 cdc24-1M. WiglerCdc42-6MATahis3 trp1-289 ura3-52 URA3::cdc42−6, GAL1p-CDC42::LEU2P. BrennwaldDBY57191MATaade2 lys2 his3 trp1 leu2 ura3 cmd1-Δ1::TRP1 ade3::HIS3::cmd 1-239A. MayerMBY 004MATahis3-Δ leu2-3,-112 lys2-Δ801 200 trp1-901 suc2-Δ9 ura3-52 gga1::HIS5spL gga2::TRP1 1H. PelhamYMK021MATahis3 leu2 trp1 ura3-52 myo2-66P. NovickMyo4ΔMATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0 myo4DEuroscarfAAY104MATaleu2-3,-112 his3-Δ 200 trp1- 901 lys2 Δ801 suc2-Δ9 pik1Δ::HIS3 carrying pRS314pik1-83 (TRP1, CEN6, pik1-83)S. EmrAAY116MATα his3-Δ200 leu2-3,-112 lys2 Δ801 trp1-901 suc2-Δ9 pik1Δ::HIS3 arf1Δ::HIS3 carrying pRS314pik1-83 (TRP1, CEN6, pik1-83)S. EmrABY1210MATα his3-200 leu2-3,112 trp1-1 ura3-52 ras2::LEU2A. BretscherBY582MATahis3-Δ200 leu2-3,112 URA3::rho3-V51 rho3Δ::LEU2P. BrennwaldH2MATα his4-580 leu2-3,112 trp1-289 ura3-52 sec1-1S. KeranenBY100MATaura3-52 his3-Δ200 LEU2::sec4-P48 sec4Δ::HIS3P. BrennwaldNY774MATα ura3-52 leu2-3,112 sec4-8P. NovickNY782MATaleu2-3,112 ura3-52 sec9-4P. NovickBY70MATα ura3-52 leu2-3,112 sec9-7P. BrennwaldNY784MATα leu2-3,112 ura3-52 sec10-2P. NovickNY786MATaleu2-3,112 ura3-52 sec15-2P. NovickVLSec22MATaleu2-3,112 ura3-52 trp1 his3 sec22-2J. GerstBY570MATα leu2-3,112 his3-Δ200 ura3-52 sro7Δ::LEU2P. BrennwaldSAY1MATα leu2-3,112 his3-Δ200 ura3-52 sro7Δ::LEU2 sro77Δ::URA3This studyH1241MATaade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 sso1-Δ1 sso2-1H. RonneABY1226MATα his3-200 leu2-3,112 trp1-1 ura3-52 tpm1::kanMXA. BretscherABY944MATα his3-Δ200 leu2-3,112 lys2-801 ura3-52 trp1-1 tpm1-2::LEU2 tpm2Δ::HIS3A. BretscherNSY348MATahis4 lys2 ura3-52 ypt31::HIS3 ypt32-A141DP. Poon Open table in a new tab Plasmids bearing genomic ASH1 were created for this study. The coding region of the ASH1 was amplified by PCR from genomic DNA using forward (Ash1-For, 5′-CCTATCGCTCCTGTCCTATCCTTATTACG-3′) and reverse (Ash1-Rev, 5′-AGTTATTAGTTGAAAGAGCTCCAGTTATCC-3′) oligonucleotides. The 1751-bp fragment was cloned into pGEM-T vector (Promega, Madison, WI) to yield pGEM-ASH1. For creation of the DNA template used to make the RNA probe for in situ hybridization, a 250-bp fragment of the ASH1 coding region was obtained by the digestion of pGEM-ASH1 with SphI and NdeI. This fragment was then ligated into the SphI and NdeI sites of pGEM to yield pGEM-ASH1short. Fluorescence in Situ Hybridization (FISH) and Immunofluorescence—ASH1 mRNA was detected by FISH with the following modifications. Cultures were grown to A600 = 0.5, fixed in 4% formaldehyde for 1 h, washed, and then spheroplasted in 100 mm potassium phosphate buffer, pH 6.5, containing 1.2 m sorbitol, 30 mm β-mercaptoethanol, and 40 μg/ml zymolase (100T; ICN Biomedicals, Aurora, OH) for 30 min at 24 °C. Spheroplasts were washed and spread onto clean polylysine-coated, multiwell test slides (ICN Biomedicals). Cells were incubated for 1 h at 50 °C in hybridization mix (50% formamide, 5× SSC, yeast tRNA (1 mg/ml), heparin (100 μg/ml), 1× Denhardt's solution, 0.1% Tween 20, 0.1% CHAPS, and 5 mm EDTA) and then incubated overnight at 50 °C in hybridization mix containing 5 μg/ml digoxigenin-labeled 250-bp ASH1 RNA probes. ASH1 RNA probes were synthesized in both the sense and antisense orientations, using the digoxigenin RNA labeling kit (Roche Applied Science) according to the manufacturer's protocol. After hybridization, cells were washed in 0.2× SSC and blocked in 1× PBS containing 0.1% Triton X-100 and 10% horse serum. Cells were incubated with horseradish peroxidase-conjugated anti-digoxigenin monoclonal antibodies (1:500) (Jackson ImmunoResearch, West Grove, PA) in blocking buffer for 2 h. After washing, cells were incubated with Cy5-conjugated anti-horseradish peroxidase antibodies (1:100) (Jackson ImmunoResearch) for 2 h at room temperature. Cells were then washed and mounted in 0.01 m Tris-Cl, pH 8.4, 90% glycerol, 1 mg/ml p-phenylenediamine, and 0.1 μg/ml propidium iodide (Sigma). The actin cytoskeleton was stained with rhodamine-conjugated phalloidin (Sigma). Cells were grown to A600 = 0.5 in YPD and fixed with formaldehyde at a final concentration of 4% for 1 h. Cells were harvested, washed twice with 1× PBS, permeabilized using 0.5% Nonidet P-40 in PBS for 5 min, and washed again with 1× PBS. For staining, 100-μl aliquots of cells were incubated with rhodamine-conjugated phalloidin (final concentration of 0.165 μm) for 15-60 min on ice and washed with 1× PBS. Cells were mixed with mounting medium and mounted on glass slides with coverslips prior to visualization. To obtain quantitative data on the organization of the actin cytoskeleton, between 50 and 100 cells of each strain were scored for the distribution of actin patches in the mother and bud. Data are summarized in Table V.Table VActin organization in yeast secretion and secretion-related mutantsStrainTemperatureExtent of actin mislocalizationMislocalization°C%WT15−018−026−037−0arf1Δ26−015+20cdc24-126−037+++65cdc42-126−033+++90cdc42-626+++6033+++90ras2Δ26++3037+++70rho3-val5126++3015+++70sec4-826+2037++40ypt31Δ26++50ypt32-137+++90sec1-126+2037++40sec9-426++3037+++80sec9-726++4037+++80sec10-226−037++50sec15-226+2037++40sec22-226−337+++89sro7Δ26−037++30sro77Δ26+20sro7Δ15+++70sso2-126−037+++85cmd1-23926+2037+++90gga1Δ26++20gga2Δ37+++80myo2-6626++3037+++70myo4Δ26−037+25pik1-8326++4037+++90 Open table in a new tab Image Analysis—Fluorescence imaging was performed using a Zeiss LSM confocal microscope with LSM510 software equipped with ×40 and ×100 oil immersion lenses. The following wavelengths were used: rhodamine-phalloidin (excitation 545 nm and emission 560-580 nm); GFP (excitation 480 nm and emission 530 nm), and Cy5 (excitation 650 nm and emission 680 nm). Control experiments for in situ hybridization showed no observable background using the sense ASH1 RNA probe. Likewise, no signal was observed in the absence of primary antibodies. For live cell imaging, cells were grown overnight to log phase in synthetic medium at 26 °C and shifted to synthetic medium lacking methionine for 1 h to induce the production of coat protein-GFP. For temperature-shift experiments, cells were incubated in medium lacking methionine at the appropriate restrictive temperature. Cells were concentrated, supplemented with gelatin (Sigma) or low melting point agarose (FMC, Rockland, ME) (0.25 or 0.5% final concentration, respectively), and mounted on glass slides or coverslips. A temperature-controlled chamber (Warner Instrument Corp.) was used to maintain cells at constant temperature. Optical sectioning was performed as described in Shaw and Quatrano (29Shaw S.L. Quatrano R.S. Development (Camb.). 1996; 122: 2623-2630PubMed Google Scholar). Cells were sectioned optically at 0.75-μm increments for a total of 3.0 μm. Measurement of mRNA Localization and Distribution in Situ—To obtain quantitative data on the localization of the ASH1 mRNA in each strain, between 30 and 80 cells with visible buds (i.e. cells in S and G2/M phase) were scored for a localized or mislocalized ASH1 mRNA signal using FISH (note: ASH1 mRNA localizes to the newly forming bud tip in early S phase). Thus, an ASH1 mRNA signal was considered as localized when it was present at the bud tip or localized along one side of the bud. An mRNA signal was considered as mislocalized to the bud and mother when it was distributed in both the bud and mother cell (i.e. transported to but not anchored at the bud tip). An mRNA signal was considered mislocalized to the mother when it was found in the mother cell only (neither transported to nor anchored in the bud). The standard deviation obtained from two separate counts (per strain) was calculated. The data obtained are summarized in Tables II, III, IV.Table IIIn situ ASH1 mRNA localization in small GTPase mutantsProteinGenotypeASH1 mRNA localization (%)°CBud tipaAnchored to distal bud tip.Bud and mother cellbMislocalized to bud and mother cell.Mother cellcMislocalized to mother cell.MislocalizeddTotal mislocalized ASH1 mRNA. (total)2676 ± 819 ± 5524WT1573 ± 321 ± 86273783 ± 713 ± 2417Cdc42cdc42-62650 ± 648 ± 92503330 ± 833 ± 337 ± 770Cdc24cdc24-12673 ± 221 ± 16273725 ± 222 ± 253 ± 275Ypt31/32ypt31Δ ypt32-A141D2662 ± 935 ± 103383730 ± 652 ± 718 ± 970Rho3rho3-V512661 ± 630 ± 49391515 ± 232 ± 653 ± 785Sec4sec4-P482680 ± 216 ± 54201532 ± 661 ± 4768sec4-82675 ± 1017 ± 28253719 ± 371 ± 510 ± 681Ras2ras2Δ2678 ± 919 ± 73223730 ± 259 ± 811 ± 370a Anchored to distal bud tip.b Mislocalized to bud and mother cell.c Mislocalized to mother cell.d Total mislocalized ASH1 mRNA. Open table in a new tab Table IIIIn situ ASH1 mRNA localization in SNARE, SNARE regulator, and exocyst mutantsProteinGenotypeASH1 mRNA localization (%)°CBud tipBud and mother cellMother cellMislocalized (total)1573 ± 321 ± 8627WTaWT, wild type.2676 ± 819 ± 55243783 ± 713 ± 2417Sro7sro7Δ2683 ± 815 ± 62173744 ± 251 ± 4556Sro77sro77Δ2687 ± 69 ± 24133775 ± 218725Sro7/77sro7Δ2666 ± 1032 ± 7234sro77Δ1521 ± 261 ± 1018 ± 779Sec10sec10-22686 ± 414 ± 50223751 ± 1044 ± 7549Sec15sec15-22678 ± 815 ± 37223747 ± 1039 ± 414 ± 553Sec1sec1-12683 ± 917 ± 60173751 ± 445 ± 6449Sso2sso2-12686 ± 214 ± 50143764 ± 428 ± 4836a WT, wild type. Open table in a new tab Table IVIn situ ASH1 mRNA localization in cytoskeleton and other mutantsProteinGenotypeASH1 mRNA localization (%)°CBud tipBud and mother cellMother cellMislocalized (total)WTaWT, wild-type background.1573 ± 321 ± 86272676 ± 819 ± 55243783 ± 713 ± 2417Tpm1tpm1Δ2668 ± 1129 ± 73323720 ± 762 ± 818 ± 680Tpm1/2tpm1-22675 ± 418 ± 5725tpm2Δ3723 ± 752 ± 425 ± 877Myo4myo4ΔWTaWT, wild-type background. 2683 ± 915 ± 7217ΔbΔ deletion. 2616 ± 572 ± 412 ± 284Myo2myo2-662681 ± 816 ± 53193759 ± 629 ± 212 ± 541Pik1pik1-832668 ± 725 ± 37323721 ± 168 ± 711 ± 679Calmodulincmd1-2392654 ± 342 ± 84463720 ± 622 ± 858 ± 580Arf1arf1Δ2663 ± 731 ± 1637Pik1pik1-833746 ± 448 ± 5654a WT, wild-type background.b Δ deletion. Open table in a new tab In Situ Localization of ASH1 mRNA in Small GTPase Mutants of the Late Secretory Pathway—ASH1 mRNA localization to the bud tip has been shown to be dependent upon proteins involved in mRNA binding and transport along the actin cytoskeleton (2Kloc M. Zearfoss N.R. Etkin L.D. Cell. 2002; 108: 533-544Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 8Long R.M. Singer R.H. Meng X. Gonzalez I. Nasmyth K. Jansen R.P. Science. 1997; 277: 383-387Crossref PubMed Scopus (416) Google Scholar, 9Takizawa P.A. Sil A. Swedlow J.R. Herskowitz I. Vale R.D. Nature. 1997; 389: 90-93Crossref PubMed Scopus (313) Google Scholar, 10Jansen R.P. FASEB J. 1999; 13: 455-466Crossref PubMed Scopus (139) Google Scholar, 11Chartrand P. Singer R.H. Long R.M. Annu. Rev. Cell Dev. Biol. 2001; 17: 297-310Crossref PubMed Scopus (62) Google Scholar, 12Jansen R.P. Dowzer C. Michaelis C. Galova M. Nasmyth K. Cell. 1996; 84: 687-697Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). The latter include actin, tropomyosins 1/2, and myosin 4, and these are encoded by ACT1, TPM1/2, and MYO4, respectively. To determine the involvement of known secretory proteins, we employed fluorescence-based in situ hybridization (FISH) by using digoxigenin-labeled sense and antisense ASH1 probes and mutants of the late secretory pathway in yeast. We first examined whether temperature-sensitive mutations in small GTPases may influence the localization of ASH1 mRNA after shifting cells to restrictive temperatures (see Table II and Fig. 1A). ASH1 mRNA localization was examined in budded S and G2/M phase yeast cells by using FISH, and the results were statistically compiled from 30 to 80 cells per condition. Specific FISH signals were observed by using the antisense probes only. 2S. Aronov and J. E. Gerst, unpublished observations. We found that >75% of wild-type cells had ASH1 mRNA present at the bud tip (Table II and note examples in Fig. 1A), as shown previously (8Long R.M. Singer R.H. Meng X. Gonzalez I. Nasmyth K. Jansen R.P. Science. 1997; 277: 383-387Crossref PubMed Scopus (416) Google Scholar, 9Takizawa P.A. Sil A. Swedlow J.R. Herskowitz I. Vale R.D. Nature. 1997; 389: 90-93Crossref PubMed Scopus (313) Google Scholar), and did not vary significantly with temperature (15-37 °C). Nonetheless, a small number of cells (<20%) were found to have some mRNA located in the nucleus of the mother cell, the latter detected by propidium iodide staining, which probably represents a maternal pool of ASH1 mRNA that had yet to be exported and delivered to the daughter cell. In contrast to wild-type cells, less than 20% of myo4Δ deletion mutants had ASH1 mRNA localized at the bud tip (Table IV and Fig. 1A). Thus, myo4Δ mutants are defective in ASH1 mRNA localization, as determined previously (8Long R.M. Singer R.H. Meng X. Gonzalez I. Nasmyth K. Jansen R.P. Science. 1997; 277: 383-387Crossref PubMed Scopus (416) Google Scholar, 9Takizawa P.A. Sil A. Swedlow J.R. Herskowitz I. Vale R.D. Nature. 1997; 389: 90-93Crossref PubMed Scopus (313) Google Scholar). Most interestingly, we found that a temperature-sensitive mutation in Cdc42 (cdc42-6), which was proposed to act only upon vesicle transport at 33 °C and not to affect the actin cytoskeleton (17Adamo J.E. Moskow J.J. Gladfelter A.S. Viterbo D. Lew D.J. Brennwald P.J. J. Cell Biol. 2001; 155: 581-592Crossref PubMed Scopus (137) Google Scholar), had prominent effects upon ASH1 mRNA localization at either permissive (26 °C) or restrictive (33 °C) temperatures (Table II and Fig. 1A). At the permissive temperature about 50% of the mRNA was mislocalized in the bud or to the mother cell, whereas 70% was mislocalized at the restrictive temperature. Moreover, nearly identical results were obtained with a temperature-sensitive mutant of the Cdc42 exchange factor, Cdc24 (cdc24-1), which showed similar amounts of mislocalized ASH1 mRNA overall (Table II). Thus, Cdc42 is likely to play a significant role in the delivery of ASH1 to the daughter cell. Next, we examined other GTPases (Table II) including the following: Sec4, which confers secretory vesicle tethering and SNARE assembly (30Guo W. Sacher M. Barrowman J. Ferro-Novick S. Novick P. Trends Cell Biol. 2000; 10: 251-255Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar); Rho3, which regulates actin assembly but may also play a role in vesicle transport (16Adamo J.E. Rossi G. Brennwald P. Mol. Biol. Cell. 1999; 12: 4121-4133Crossref Scopus (174) Google Scholar); Ypt31/32, which is involved in Golgi export (31Jedd G. Mulholland J. Segev N. J. Cell Biol. 1997; 137: 563-580Crossref PubMed Scopus (180) Google Scholar); and Ras2, which regulates signaling pathways involved in both cell growth and actin regulation (32Ho J. Bretscher A. Mol. Biol. Cell. 2001; 12: 1541-1555Crossref PubMed Scopus (59) Google Scholar). Similar to cdc42-6, we found that an allele of Rho3 (rho3-V51) that was proposed to act upon vesicle transport alone (16Adamo J.E. Rossi G. Brennwald P. Mol. Biol. Cell. 1999; 12: 4121-4133Crossref Scopus (174) Google Scholar) had strong effects upon ASH1 mRNA localization (Table II). At the permissive temperature (26 °C) about 40% of the cells had mislocalized ASH1 mRNA, whereas this increased to 85% at the restrictive temperature (15 °C; 1 h), wherein most of the mRNA accumulated exclusively in the mother cell. Interestingly, ASH1 mRNA was mislocalized at restrictive temperatures with all of the other GTPase mutants examined, including the following: sec4-8 (37 °C; 1 h) (for example, see Fig. 1A); sec4-P48 (15 °C; 1 h); ypt31Δ ypt32-1 (37 °C; 1 h); and ras2Δ cells (37 °C; 1 h). However, in these particular strains ASH1 mRNA was distributed in both mother and daughter cells and was not restricted to the mother cell as seen in cdc42-6 and rho3-V51 cells. Nonetheless, the finding that two different alleles of SEC4, which encodes a protein involved in the terminal steps of vesicle docking and fusion, affect ASH1 mRNA localization strongly suggested a direct involvement of the secretory pathway in mRNA transport and localization. In Situ Localization of ASH1 mRNA in Exocyst, SNARE Regulator, and SNARE Mutants of the Late Secretory Pathway—We next examined ASH1 mRNA localization in situ in mutants of proteins directly involved in vesicle tethering, docking, and fusion (Tabl" @default.
• W2122724972 created "2016-06-24" @default.
• W2122724972 creator A5042969862 @default.
• W2122724972 creator A5069272617 @default.
• W2122724972 date "2004-08-01" @default.
• W2122724972 modified "2023-09-30" @default.
• W2122724972 title "Involvement of the Late Secretory Pathway in Actin Regulation and mRNA Transport in Yeast" @default.
• W2122724972 cites W1508614407 @default.
• W2122724972 cites W1526675316 @default.
• W2122724972 cites W1699722243 @default.
• W2122724972 cites W1932699125 @default.
• W2122724972 cites W1936931129 @default.
• W2122724972 cites W197316475 @default.
• W2122724972 cites W1974985862 @default.
• W2122724972 cites W1981990755 @default.
• W2122724972 cites W1998418805 @default.
• W2122724972 cites W2003190277 @default.
• W2122724972 cites W2007391251 @default.
• W2122724972 cites W2013821673 @default.
• W2122724972 cites W2014715442 @default.
• W2122724972 cites W2020809039 @default.
• W2122724972 cites W2031408226 @default.
• W2122724972 cites W2034884676 @default.
• W2122724972 cites W2038475514 @default.
• W2122724972 cites W2039701501 @default.
• W2122724972 cites W2040169742 @default.
• W2122724972 cites W2042903750 @default.
• W2122724972 cites W2045243735 @default.
• W2122724972 cites W2057163724 @default.
• W2122724972 cites W2060006253 @default.
• W2122724972 cites W2074622507 @default.
• W2122724972 cites W2080273350 @default.
• W2122724972 cites W2086442201 @default.
• W2122724972 cites W2090583357 @default.
• W2122724972 cites W2092154357 @default.
• W2122724972 cites W2093081532 @default.
• W2122724972 cites W2098440355 @default.
• W2122724972 cites W2099269199 @default.
• W2122724972 cites W2106881932 @default.
• W2122724972 cites W2109672926 @default.
• W2122724972 cites W2111301572 @default.
• W2122724972 cites W2113945036 @default.
• W2122724972 cites W2118356782 @default.
• W2122724972 cites W2122322808 @default.
• W2122724972 cites W2123799523 @default.
• W2122724972 cites W2127540003 @default.
• W2122724972 cites W2135563458 @default.
• W2122724972 cites W2136178963 @default.
• W2122724972 cites W2160524202 @default.
• W2122724972 cites W2170280114 @default.
• W2122724972 cites W322031617 @default.
• W2122724972 doi "https://doi.org/10.1074/jbc.m402068200" @default.
• W2122724972 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15192110" @default.
• W2122724972 hasPublicationYear "2004" @default.
• W2122724972 type Work @default.
• W2122724972 sameAs 2122724972 @default.
• W2122724972 citedByCount "50" @default.
• W2122724972 countsByYear W21227249722013 @default.
• W2122724972 countsByYear W21227249722014 @default.
• W2122724972 countsByYear W21227249722015 @default.
• W2122724972 countsByYear W21227249722016 @default.
• W2122724972 countsByYear W21227249722017 @default.
• W2122724972 countsByYear W21227249722020 @default.
• W2122724972 countsByYear W21227249722021 @default.
• W2122724972 countsByYear W21227249722022 @default.
• W2122724972 crossrefType "journal-article" @default.
• W2122724972 hasAuthorship W2122724972A5042969862 @default.
• W2122724972 hasAuthorship W2122724972A5069272617 @default.
• W2122724972 hasBestOaLocation W21227249721 @default.
• W2122724972 hasConcept C104317684 @default.
• W2122724972 hasConcept C105580179 @default.
• W2122724972 hasConcept C119062480 @default.
• W2122724972 hasConcept C125705527 @default.
• W2122724972 hasConcept C158617107 @default.
• W2122724972 hasConcept C185592680 @default.
• W2122724972 hasConcept C2779222958 @default.
• W2122724972 hasConcept C55493867 @default.
• W2122724972 hasConcept C6492254 @default.
• W2122724972 hasConcept C86803240 @default.
• W2122724972 hasConcept C95444343 @default.
• W2122724972 hasConceptScore W2122724972C104317684 @default.
• W2122724972 hasConceptScore W2122724972C105580179 @default.
• W2122724972 hasConceptScore W2122724972C119062480 @default.
• W2122724972 hasConceptScore W2122724972C125705527 @default.
• W2122724972 hasConceptScore W2122724972C158617107 @default.
• W2122724972 hasConceptScore W2122724972C185592680 @default.
• W2122724972 hasConceptScore W2122724972C2779222958 @default.
• W2122724972 hasConceptScore W2122724972C55493867 @default.
• W2122724972 hasConceptScore W2122724972C6492254 @default.
• W2122724972 hasConceptScore W2122724972C86803240 @default.
• W2122724972 hasConceptScore W2122724972C95444343 @default.
• W2122724972 hasIssue "35" @default.
• W2122724972 hasLocation W21227249721 @default.
• W2122724972 hasOpenAccess W2122724972 @default.
• W2122724972 hasPrimaryLocation W21227249721 @default.
• W2122724972 hasRelatedWork W2029141018 @default.
• W2122724972 hasRelatedWork W2039005185 @default.
• W2122724972 hasRelatedWork W2046185360 @default.

• next