Summary.
The formation of actin filaments is crucial for endocytosis and other interrelated cellular phenomena such as motility, polarized morphogenesis, and cytokinesis. In this paper we have investigated the role of the WASP/Las17-interacting protein Bzz1p in endocytosis and trafficking to the vacuole. We and others have recently shown that Bzz1p is an actin patch protein that interacts directly with Las17p via a SH3-polyproline interaction. Bzz1p functions with type I myosins to restore polarity of the actin cytoskeleton after NaCl stress. In an in vitro bead assay, GST-Bzz1p fusion protein triggers a functional actin polymerization machinery through its two C-terminal SH3 domains. In this paper we implicate Bzz1p with the type I myosins both in fluid-phase and in the internalization step of receptor-mediated endocytosis. As deduced from their localization as GFP fusions, the vacuolar delivery of endocytic and biosynthetic cargoes as well as the multivesicular body pathway appear unaffected. We further elucidate Bzz1p direct participation in actin polymerization by demonstrating that each of the SH3 domains of Bzz1p individually is able to trigger actin polymerization in a cell-free system dependent on Arp2/3, Las17p, Vrp1p, and the type I myosins. Taken together, our results show that Bzz1p participates, essentially via its SH3 domains, in early steps of endocytosis together with known actin nucleation activators.
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References
BL Anderson I Boldogh M Evangelista C Boone LA Greene LA Pon (1998) ArticleTitleThe Src homology domain 3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of actin polarization. J Cell Biol 141 1357–1370 Occurrence Handle10.1083/jcb.141.6.1357 Occurrence Handle1:CAS:528:DyaK1cXjvFKksbs%3D Occurrence Handle9628892
CJ Bonangelino EM Chavez JS Bonifacino (2002) ArticleTitleGenomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae. Mol Biol Cell 13 2486–2501 Occurrence Handle10.1091/mbc.02-01-0005 Occurrence Handle1:CAS:528:DC%2BD38XlvVCku7k%3D Occurrence Handle12134085
FS Chang CJ Stefan KJ Blumer (2003) ArticleTitleA WASp homolog powers actin polymerization-dependent motility of endosomes in vivo. Curr Biol 13 455–463 Occurrence Handle1:CAS:528:DC%2BD3sXit12ntbY%3D Occurrence Handle12646127
E Conibear TH Stevens (2000) ArticleTitleVps52p, Vps53p, and Vps54p form a novel multisubunit complex required for protein sorting at the yeast late Golgi. Mol Biol Cell 11 305–323 Occurrence Handle1:CAS:528:DC%2BD3cXosFyitw%3D%3D Occurrence Handle10637310
T Darsow CG Burd SD Emr (1998) ArticleTitleAcidic di-leucine motif essential for AP-3-dependent sorting and restriction of the functional specificity of the Vam3p vacuolar t-SNARE. J Cell Biol 142 913–922 Occurrence Handle10.1083/jcb.142.4.913 Occurrence Handle1:CAS:528:DyaK1cXls1eitbw%3D Occurrence Handle9722605
Dulic V, Egerton M, Elguindi I, Raths S, Singer B, Riezman H (1991) Yeast endocytosis assays. In: Guthrie C (ed) Guide to yeast genetics and molecular cell biology, part A. Academic Press, London, pp 697–710 (Methods in enzymology, vol 194)
A Eugster EI Pecheur F Michel B Winsor F Letourneur S Friant (2004) ArticleTitleEnt5p is required with Ent3p and Vps27p for ubiquitin-dependent protein sorting into the multivesicular body. Mol Biol Cell 15 3031–3041 Occurrence Handle10.1091/mbc.E03-11-0793 Occurrence Handle1:CAS:528:DC%2BD2cXlslWnsrg%3D Occurrence Handle15107463
M Evangelista BM Klebl AH Tong BA Webb T Leeuw E Leberer M Whiteway DY Thomas C Boone (2000) ArticleTitleA role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex. J Cell Biol 148 353–362 Occurrence Handle10.1083/jcb.148.2.353 Occurrence Handle1:CAS:528:DC%2BD3cXot1eqsg%3D%3D Occurrence Handle10648568
– Pruyne D, Amberg DC, Boone C, Bretscher A (2002) Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast. Nat Cell Biol 4: 32–41
S Friant EI Pecheur A Eugster F Michel Y Lefkir D Nourrisson F Letourneur (2003) ArticleTitleEnt3p is a PtdIns(3,5)P2 effector required for protein sorting to the multivesicular body. Dev Cell 5 499–511 Occurrence Handle10.1016/S1534-5807(03)00238-7 Occurrence Handle1:CAS:528:DC%2BD3sXnsFKltrk%3D Occurrence Handle12967568
JM Galan V Moreau B Andre C Volland R Haguenauer-Tsapis (1996) ArticleTitleUbiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease. J Biol Chem 271 10946–10952 Occurrence Handle1:CAS:528:DyaK28XivVaqu7c%3D Occurrence Handle8631913
MI Geli H Riezman (1996) ArticleTitleRole of type I myosins in receptor-mediated endocytosis in yeast. Science 272 533–535 Occurrence Handle1:CAS:528:DyaK28XisFOmsbk%3D Occurrence Handle8614799
– Lombardi R, Schmelzl B, Riezman H (2000) An intact SH3 domain is required for myosin I-induced actin polymerization. EMBO J 19: 4281–4291
HV Goodson BL Anderson HM Warrick LA Pon JA Spudich (1996) ArticleTitleSynthetic lethality screen identifies a novel yeast myosin I gene (MYO5): myosin I proteins are required for polarization of the actin cytoskeleton. J Cell Biol 133 1277–1291 Occurrence Handle10.1083/jcb.133.6.1277 Occurrence Handle1:CAS:528:DyaK28XjslKjtr4%3D Occurrence Handle8682864
CW Gourlay H Dewar DT Warren R Costa N Satish KR Ayscough (2003) ArticleTitleAn interaction between Sla1p and Sla2p plays a role in regulating actin dynamics and endocytosis in budding yeast. J Cell Sci 116 2551–2564 Occurrence Handle10.1242/jcs.00454 Occurrence Handle1:CAS:528:DC%2BD3sXltFemtrs%3D Occurrence Handle12734398
L Hicke H Riezman (1996) ArticleTitleUbiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell 84 277–287 Occurrence Handle10.1016/S0092-8674(00)80982-4 Occurrence Handle1:CAS:528:DyaK28Xnt1egtg%3D%3D Occurrence Handle8565073
– Zanolari B, Riezman H (1998) Cytoplasmic tail phosphorylation of the alpha-factor receptor is required for its ubiquitination and internalization. J Cell Biol 141: 349–358
HN Higgs TD Pollard (2001) ArticleTitleRegulation of actin filament network formation through ARP2/3 complex: activation by a diverse array of proteins. Annu Rev Biochem 70 649–676 Occurrence Handle10.1146/annurev.biochem.70.1.649 Occurrence Handle1:CAS:528:DC%2BD3MXlsVehtLs%3D Occurrence Handle11395419
GA Jonsdottir R Li (2004) ArticleTitleDynamics of yeast myosin I: evidence for a possible role in scission of endocytic vesicles. Curr Biol 14 1604–1609 Occurrence Handle10.1016/j.cub.2004.08.055 Occurrence Handle1:CAS:528:DC%2BD2cXntleks7k%3D Occurrence Handle15341750
G Jung K Remmert X Wu JM Volosky JA Hammer Suffix3rd (2001) ArticleTitleThe Dictyostelium CARMIL protein links capping protein and the Arp2/3 complex to type I myosins through their SH3 domains. J Cell Biol 153 1479–1497 Occurrence Handle10.1083/jcb.153.7.1479 Occurrence Handle1:CAS:528:DC%2BD3MXkslensb8%3D Occurrence Handle11425877
M Kaksonen Y Sun DG Drubin (2003) ArticleTitleA pathway for association of receptors, adaptors, and actin during endocytic internalization. Cell 115 475–487 Occurrence Handle10.1016/S0092-8674(03)00883-3 Occurrence Handle1:CAS:528:DC%2BD3sXpt1Oqsrw%3D Occurrence Handle14622601
JV Kilmartin AE Adams (1984) ArticleTitleStructural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J Cell Biol 98 922–933 Occurrence Handle10.1083/jcb.98.3.922 Occurrence Handle1:CAS:528:DyaL2cXhsFemsbo%3D Occurrence Handle6365930
E Kubler H Riezman (1993) ArticleTitleActin and fimbrin are required for the internalization step of endocytosis in yeast. EMBO J 12 2855–2862 Occurrence Handle1:STN:280:ByyA38jnslE%3D Occurrence Handle8335001
T Lechler A Shevchenko R Li (2000) ArticleTitleDirect involvement of yeast type I myosins in Cdc42-dependent actin polymerization. J Cell Biol 148 363–373 Occurrence Handle10.1083/jcb.148.2.363 Occurrence Handle1:CAS:528:DC%2BD3cXot1eqsw%3D%3D Occurrence Handle10648569
– Jonsdottir GA, Klee SK, Pellman D, Li R (2001) A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast. J Cell Biol 155: 261–270
WL Lee M Bezanilla TD Pollard (2000) ArticleTitleFission yeast myosin-I, Myo1p, stimulates actin assembly by Arp2/3 complex and shares functions with WASp. J Cell Biol 151 789–800 Occurrence Handle10.1083/jcb.151.4.789 Occurrence Handle1:CAS:528:DC%2BD3cXotFens70%3D Occurrence Handle11076964
SK Lemmon LM Traub (2000) ArticleTitleSorting in the endosomal system in yeast and animal cells. Curr Opin Cell Biol 12 457–466 Occurrence Handle10.1016/S0955-0674(00)00117-4 Occurrence Handle1:CAS:528:DC%2BD3cXlt1Srur8%3D Occurrence Handle10873832
R Li (1997) ArticleTitleBee1, a yeast protein with homology to Wiscott-Aldrich syndrome protein, is critical for the assembly of cortical actin cytoskeleton. J Cell Biol 136 649–658 Occurrence Handle10.1083/jcb.136.3.649 Occurrence Handle1:CAS:528:DyaK2sXhtFCltrg%3D Occurrence Handle9024694
J Lippincott R Li (2000) ArticleTitleInvolvement of PCH family proteins in cytokinesis and actin distribution. Microsc Res Tech 49 168–172 Occurrence Handle10.1002/(SICI)1097-0029(20000415)49:2<168::AID-JEMT9>3.0.CO;2-T Occurrence Handle1:CAS:528:DC%2BD3cXjslSltL8%3D Occurrence Handle10816256
A Madania P Dumoulin S Grava H Kitamoto C Scharer-Brodbeck A Soulard V Moreau B Winsor (1999) ArticleTitleThe Saccharomyces cerevisiae homologue of human Wiskott-Aldrich syndrome protein Las17p interacts with the Arp2/3 complex. Mol Biol Cell 10 3521–3538 Occurrence Handle1:CAS:528:DyaK1MXmvFaitro%3D Occurrence Handle10512884
BJ Mayer (2001) ArticleTitleSH3 domains: complexity in moderation. J Cell Sci 114 1253–1263 Occurrence Handle1:CAS:528:DC%2BD3MXjt1Omt7k%3D Occurrence Handle11256992
CJ Merrifield ME Feldman L Wan W Almers (2002) ArticleTitleImaging actin and dynamin recruitment during invagination of single clathrin-coated pits. Nat Cell Biol 4 691–698 Occurrence Handle10.1038/ncb837 Occurrence Handle1:CAS:528:DC%2BD38Xms1aht7s%3D Occurrence Handle12198492
V Moreau A Madania RP Martin B Winsor (1996) ArticleTitleThe Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton. J Cell Biol 134 117–132 Occurrence Handle10.1083/jcb.134.1.117 Occurrence Handle1:CAS:528:DyaK28XktVCqur4%3D Occurrence Handle8698808
– Galan JM, Devilliers G, Haguenauer-Tsapis R, Winsor B (1997) The yeast actin-related protein Arp2p is required for the internalization step of endocytosis. Mol Biol Cell 8: 1361–1375
AL Munn BJ Stevenson MI Geli H Riezman (1995) ArticleTitleend5, end6, and end7: mutations that cause actin delocalization and block the internalization step of endocytosis in Saccharomyces cerevisiae. Mol Biol Cell 6 1721–1742 Occurrence Handle1:CAS:528:DyaK2MXpvVWntbw%3D Occurrence Handle8590801
SN Naqvi R Zahn DA Mitchell BJ Stevenson AL Munn (1998) ArticleTitleThe WASp homologue Las17p functions with the WIP homologue End5p/verprolin and is essential for endocytosis in yeast. Curr Biol 8 959–962 Occurrence Handle10.1016/S0960-9822(98)70396-3 Occurrence Handle1:CAS:528:DyaK1cXlvFajtbg%3D Occurrence Handle9742397
EM Ostap P Maupin SK Doberstein IC Baines ED Korn TD Pollard (2003) ArticleTitleDynamic localization of myosin-I to endocytic structures in Acanthamoeba. Cell Motil Cytoskeleton 54 29–40 Occurrence Handle10.1002/cm.10081 Occurrence Handle1:CAS:528:DC%2BD3sXhtVSgtbY%3D Occurrence Handle12451593
D Pruyne M Evangelista C Yang E Bi S Zigmond A Bretscher C Boone (2002) ArticleTitleRole of formins in actin assembly: nucleation and barbed-end association. Science 297 612–615 Occurrence Handle10.1126/science.1072309 Occurrence Handle1:CAS:528:DC%2BD38XlslWrs7c%3D Occurrence Handle12052901
C Raiborg TE Rusten H Stenmark (2003) ArticleTitleProtein sorting into multivesicular endosomes. Curr Opin Cell Biol 15 446–455 Occurrence Handle10.1016/S0955-0674(03)00080-2 Occurrence Handle1:CAS:528:DC%2BD3sXlvVGjtr0%3D Occurrence Handle12892785
F Reggiori HR Pelham (2001) ArticleTitleSorting of proteins into multivesicular bodies: ubiquitin-dependent and -independent targeting. EMBO J 20 5176–5186 Occurrence Handle10.1093/emboj/20.18.5176 Occurrence Handle1:CAS:528:DC%2BD3MXns1Ghsrc%3D Occurrence Handle11566881
H Riezman A Munn MI Geli L Hicke (1996) ArticleTitleActin-, myosin- and ubiquitin-dependent endocytosis. Experientia 52 1033–1041 Occurrence Handle10.1007/BF01952099 Occurrence Handle1:STN:280:ByiC2c7pvFc%3D Occurrence Handle8988243
AA Rodal AL Manning BL Goode DG Drubin (2003) ArticleTitleNegative regulation of yeast WASp by two SH3 domain-containing proteins. Curr Biol 13 1000–1008 Occurrence Handle10.1016/S0960-9822(03)00383-X Occurrence Handle1:CAS:528:DC%2BD3sXkvVKgt70%3D Occurrence Handle12814545
R Rohatgi P Nollau HY Ho MW Kirschner BJ Mayer (2001) ArticleTitleNck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway. J Biol Chem 276 26448–26452 Occurrence Handle10.1074/jbc.M103856200 Occurrence Handle1:CAS:528:DC%2BD3MXlsVKnt70%3D Occurrence Handle11340081
I Sagot SK Klee D Pellman (2002) ArticleTitleYeast formins regulate cell polarity by controlling the assembly of actin cables. Nat Cell Biol 4 42–50 Occurrence Handle1:CAS:528:DC%2BD38XhtFGlsLc%3D Occurrence Handle11740491
MG Smith SR Swamy LA Pon (2001) ArticleTitleThe life cycle of actin patches in mating yeast. J Cell Sci 114 1505–1513 Occurrence Handle1:CAS:528:DC%2BD3MXjsVyiu7g%3D Occurrence Handle11282026
T Soldati (2003) ArticleTitleUnconventional myosins, actin dynamics and endocytosis: a menage a trois? Traffic 4 358–366 Occurrence Handle10.1034/j.1600-0854.2003.t01-1-00095.x Occurrence Handle1:CAS:528:DC%2BD3sXkt1Smt7s%3D Occurrence Handle12753645
A Soulard T Lechler V Spiridonov A Shevchenko R Li B Winsor (2002) ArticleTitleSaccharomyces cerevisiae Bzz1p is implicated with type I myosins in actin patch polarization and is able to recruit actin-polymerizing machinery in vitro. Mol Cell Biol 22 7889–7906 Occurrence Handle10.1128/MCB.22.22.7889-7906.2002 Occurrence Handle1:CAS:528:DC%2BD38XotlCgsL4%3D Occurrence Handle12391157
CJ Stefan KJ Blumer (1999) ArticleTitleA syntaxin homolog encoded by VAM3 mediates down-regulation of a yeast G protein-coupled receptor. J Biol Chem 274 1835–1841 Occurrence Handle10.1074/jbc.274.3.1835 Occurrence Handle1:CAS:528:DyaK1MXpsFOmuw%3D%3D Occurrence Handle9880567
JA Swanson MT Johnson K Beningo P Post M Mooseker N Araki (1999) ArticleTitleA contractile activity that closes phagosomes in macrophages. J Cell Sci 112 307–316 Occurrence Handle1:CAS:528:DyaK1MXhsVaqsrY%3D Occurrence Handle9885284
J Taunton BA Rowning ML Coughlin M Wu RT Moon TJ Mitchison CA Larabell (2000) ArticleTitleActin-dependent propulsion of endosomes and lysosomes by recruitment of N-WASP. J Cell Biol 148 519–530 Occurrence Handle10.1083/jcb.148.3.519 Occurrence Handle1:CAS:528:DC%2BD3cXhtFSqt78%3D Occurrence Handle10662777
N Tolliday N Bouquin R Li (2001) ArticleTitleAssembly and regulation of the cytokinetic apparatus in budding yeast. Curr Opin Microbiol 4 690–695 Occurrence Handle10.1016/S1369-5274(01)00270-3 Occurrence Handle1:CAS:528:DC%2BD3MXos1Kltrc%3D Occurrence Handle11731321
AH Tong B Drees G Nardelli GD Bader B Brannetti L Castagnoli M Evangelista S Ferracuti B Nelson S Paoluzi M Quondam A Zucconi CW Hogue S Fields C Boone G Cesareni (2002) ArticleTitleA combined experimental and computational strategy to define protein interaction networks for peptide recognition modules. Science 295 321–324 Occurrence Handle10.1126/science.1064987 Occurrence Handle1:CAS:528:DC%2BD38Xkt1Kmsg%3D%3D Occurrence Handle11743162
TA Vida SD Emr (1995) ArticleTitleA new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128 779–792 Occurrence Handle10.1083/jcb.128.5.779 Occurrence Handle1:CAS:528:DyaK2MXktVGqtLw%3D Occurrence Handle7533169
DT Warren PD Andrews CW Gourlay KR Ayscough (2002) ArticleTitleSla1p couples the yeast endocytic machinery to proteins regulating actin dynamics. J Cell Sci 115 1703–1715 Occurrence Handle1:CAS:528:DC%2BD38XjslWiu7g%3D Occurrence Handle11950888
B Winsor E Schiebel (1997) ArticleTitleReview: an overview of the Saccharomyces cerevisiae microtubule and microfilament cytoskeleton. Yeast 13 399–434 Occurrence Handle10.1002/(SICI)1097-0061(199704)13:5<399::AID-YEA126>3.0.CO;2-9 Occurrence Handle1:CAS:528:DyaK2sXjtVCls74%3D Occurrence Handle9153752
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Correspondence and reprints: Equipe Cytosquelette et Trafic Intracellulaire, UMR7156 du CNRS, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue Descartes, 67084 Strasbourg, France.
Present address: Division of Biochemistry, Biozentrum, University of Basel, Basel, Switzerland.
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Soulard, A., Friant, S., Fitterer, C. et al. The WASP/Las17p-interacting protein Bzz1p functions with Myo5p in an early stage of endocytosis. Protoplasma 226, 89–101 (2005). https://doi.org/10.1007/s00709-005-0108-4
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DOI: https://doi.org/10.1007/s00709-005-0108-4