Skip to main content

Requirements of Slm proteins for proper eisosome organization, endocytic trafficking and recycling in the yeast Saccharomyces cerevisiae

Journal of Biosciences volume 36, pages 79–96 (2011)Cite this article

Abstract

Eisosomes are large immobile assemblies at the cortex of a cell under the membrane compartment of Can1 (MCC) in yeast. Slm1 has recently been identified as an MCC component that acts downstream of Mss4 in a pathway that regulates actin cytoskeleton organization in response to stress. In this study, we showed that inactivation of Slm proteins disrupts proper localization of the primary eisosome marker Pil1, providing evidence that Slm proteins play a role in eisosome organization. Furthermore, we found that slm ts mutant cells exhibit actin defects in both the ability to polarize cortical F-actin and the formation of cytoplasmic actin cables even at the permissive temperature (30°C). We further demonstrated that the actin defect accounts for the slow traffic of FM4-64-labelled endosome in the cytoplasm, supporting the notion that intact actin is essential for endosome trafficking. However, our real-time microscopic analysis of Abp1-RFP revealed that the actin defect in slm ts cells was not accompanied by a noticeable defect in actin patch internalization during receptor-mediated endocytosis. In addition, we found that slm ts cells displayed impaired membrane recycling and that recycling occurred in an actin-independent manner. Our data provide evidence for the requirement of Slm proteins in eisosome organization and endosome trafficking and recycling.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price includes VAT (Canada)

Figure 1.
Figure 2.
Figure 3
Figure 4.
Figure 5.
Figure 6
Figure 7.

Abbreviations

FITC:

fluorescein isothiocyanate

LCB:

long-chain bases

LY:

Lucifer yellow

MCC:

membrane compartment of Can1

MCP:

membrane compartment of Pma1

NA:

numerical aperture

PH:

pleckstrin homology

RME:

receptor-mediated endocytosis

YPD:

yeast peptone dextrose

References

  • Abe M, Qadota H, Hirata A and Ohya Y 2003 Lack of GTP-bound Rho1p in secretory vesicles of Saccharomyces cerevisiae. J. Cell Biol. 162 85–97

    Article  PubMed  CAS  Google Scholar 

  • Audhya A and Emr SD 2002 Stt4 PI 4-kinase localizes to the plasma membrane and functions in the Pkc1-mediated MAP kinase cascade. Dev. Cell 2 593–605

    Article  PubMed  CAS  Google Scholar 

  • Audhya A and Emr SD 2003 Regulation of PI4,5P2 synthesis by nuclear-cytoplasmic shuttling of the Mss4 lipid kinase. EMBO J. 22 4223–4236

    Article  PubMed  CAS  Google Scholar 

  • Audhya A, Loewith R, Parsons AB, Gao L, Tabuchi, M, Zhou H, Boone C, Hall MN and Emr SD 2004 Genome-wide lethality screen identifies new PI4,5P2 effectors that regulate the actin cytoskeleton. EMBO J. 23 3747–3757

    Article  PubMed  CAS  Google Scholar 

  • Balasubramanian MK, Bi E and Glotzer M 2004 Comparative analysis of cytokinesis in budding yeast, fission yeast and animal cells. Curr. Biol . 14 R806–818

    Article  PubMed  CAS  Google Scholar 

  • Betz WJ, Mao F and Smith CB 1996 Imaging exocytosis and endocytosis. Curr. Opin. Neurobiol. 6 365–371

    Article  PubMed  CAS  Google Scholar 

  • Blondel MO, Morvan J, Dupre S, Urban-Grimal D, Haguenauer-Tsapis R and Volland C 2004 Direct sorting of the yeast uracil permease to the endosomal system is controlled by uracil binding and Rsp5p-dependent ubiquitylation. Mol. Biol. Cell 15 883–895

    Article  PubMed  CAS  Google Scholar 

  • Bultynck G, Heath VL, Majeed AP, Galan JM, Haguenauer-Tsapis R and Cyert MS 2006 Slm1 and slm2 are novel substrates of the calcineurin phosphatase required for heat stress-induced endocytosis of the yeast uracil permease. Mol. Cell. Biol. 26 4729–4745

    Article  PubMed  CAS  Google Scholar 

  • Carlsson AE, Shah AD, Elking D, Karpova TS and Cooper JA 2002 Quantitative analysis of actin patch movement in yeast. Biophys. J. 82 2333–2343

    Article  PubMed  CAS  Google Scholar 

  • Chang FS, Stefan CJ and Blumer KJ 2003 A WASp homolog powers actin polymerization-dependent motility of endosomes in vivo. Curr. Biol. 13 455–463

    Article  PubMed  CAS  Google Scholar 

  • Chevallier MR 1982 Cloning and transcriptional control of a eucaryotic permease gene. Mol. Cell. Biol. 2 977–984

    PubMed  CAS  Google Scholar 

  • Desrivieres S, Cooke FT Parker PJ and Hall MN 1998 MSS4, a phosphatidylinositol-4-phosphate 5-kinase required for organization of the actin cytoskeleton in Saccharomyces cerevisiae. J. Biol. Chem. 273 15787–15793

    Article  PubMed  CAS  Google Scholar 

  • Dickson RC 2008 Thematic review series: sphingolipids. New insights into sphingolipid metabolism and function in budding yeast. J. Lipid Res. 49 909–921

    Article  PubMed  CAS  Google Scholar 

  • Drubin DG, Kaksonen M, Toret C and Sun Y 2005 Cytoskeletal networks and pathways involved in endocytosis. Novartis Found. Symp. 269 35–42; discussion 43–36, 223–230

    Article  PubMed  CAS  Google Scholar 

  • Dulic V, Egerton M, Elguindi I, Raths S, Singer B and Riezman H 1991 Yeast endocytosis assays. Methods Enzymol. 194 697–710

    Article  PubMed  CAS  Google Scholar 

  • Evangelista M, Pruyne D, Amberg DC, Boone C and Bretscher A 2002 Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast. Nat. Cell Biol. 4 260–269

    Article  PubMed  CAS  Google Scholar 

  • Fadri M, Daquinag A, Wang S, Xue T and Kunz J 2005 The pleckstrin homology domain proteins Slm1 and Slm2 are required for actin cytoskeleton organization in yeast and bind phosphatidylinositol-4,5-bisphosphate and TORC2. Mol. Biol. Cell 16 1883–1900

    Article  PubMed  CAS  Google Scholar 

  • Fischer-Parton S, Parton RM, Hickey PC, Dijksterhuis J, Atkinson HA and Read ND 2000 Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae. J. Microsc. 198 246–259

    Article  PubMed  CAS  Google Scholar 

  • Frohlich F, Moreira K, Aguilar PS, Hubner NC, Mann M, Walter P and Walther TC 2009 A genome-wide screen for genes affecting eisosomes reveals Nce102 function in sphingolipid signaling. J. Cell Biol. 185 1227–1242

    Article  PubMed  Google Scholar 

  • Galan JM, Moreau V, Andre B, Volland C and Haguenauer-Tsapis R 1996 Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease. J. Biol. Chem. 271 10946–10952

    Article  PubMed  CAS  Google Scholar 

  • Goode BL and Rodal AA 2001 Modular complexes that regulate actin assembly in budding yeast. Curr. Opin. Microbiol. 4 703–712

    Article  PubMed  CAS  Google Scholar 

  • Grossmann G, Malinsky J, Stahlschmidt W, Loibl M, Weig-Meckl I, Frommer WB, Opekarova M and Tanner W 2008 Plasma membrane microdomains regulate turnover of transport proteins in yeast. J. Cell Biol. 183 1075–1088

    Article  PubMed  CAS  Google Scholar 

  • Grossmann G, Opekarova M, Malinsky J, Weig-Meckl I and Tanner W 2007 Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast. EMBO J. 26 1–8

    Article  PubMed  CAS  Google Scholar 

  • Homma K, Terui S, Minemura M, Qadota H, Anraku Y, Kanaho Y and Ohya Y 1998 Phosphatidylinositol-4-phosphate 5-kinase localized on the plasma membrane is essential for yeast cell morphogenesis. J. Biol. Chem. 273 15779–15786

    Article  PubMed  CAS  Google Scholar 

  • Huckaba TM, Gay AC, Pantalena LF, Yang HC and Pon LA 2004 Live cell imaging of the assembly, disassembly, and actin cable-dependent movement of endosomes and actin patches in the budding yeast, Saccharomyces cerevisiae. J. Cell Biol. 167 519–530

    Article  CAS  Google Scholar 

  • Kaksonen M, Sun Y and Drubin DG 2003 A pathway for association of receptors, adaptors, and actin during endocytic internalization. Cell 115 475–487

    Article  PubMed  CAS  Google Scholar 

  • Kaksonen M, Toret CP and Drubin DG 2005 A modular design for the clathrin- and actin-mediated endocytosis machinery. Cell 123 305–320

    Article  PubMed  CAS  Google Scholar 

  • Kim K, Galletta BJ, Schmidt KO, Chang FS, Blumer KJ and Cooper JA 2006 Actin-based motility during endocytosis in budding yeast. Mol. Biol. Cell 17 1354–1363

    Article  PubMed  CAS  Google Scholar 

  • Kim K, Yamashita A, Wear MA, Maeda Y and Cooper JA 2004 Capping protein binding to actin in yeast: biochemical mechanism and physiological relevance. J. Cell Biol. 164 567–580

    Article  PubMed  CAS  Google Scholar 

  • Kurtz JE, Exinger F, Erbs P and Jund R 1999 New insights into the pyrimidine salvage pathway of Saccharomyces cerevisiae: requirement of six genes for cytidine metabolism. Curr. Gen. 36 130–136

    Article  CAS  Google Scholar 

  • Lewis MJ, Nichols BJ, Prescianotto-Baschong C, Riezman H and Pelham HR 2000 Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes. Mol. Biol. Cell 11 23–38

    PubMed  CAS  Google Scholar 

  • Lipatova Z, Tokarev AA, Jin Y, Mulholland J, Weisman LS and Segev N 2008 Direct interaction between a myosin V motor and the Rab GTPases Ypt31/32 is required for polarized secretion. Mol. Biol. Cell 19 4177–4187

    Article  PubMed  CAS  Google Scholar 

  • Liu J, Sun Y, Drubin DG and Oster GF 2009 The mechanochemistry of endocytosis. PLoS Biol. 7 e1000204

    Article  PubMed  Google Scholar 

  • Longtine MS, Mckenzie A 3rd, Demarini DJ, Shah NG, Wach A, Brachat A, Philippsen P and Pringle JR 1998 Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast (Chichester, England) 14 953–961

    Article  CAS  Google Scholar 

  • Luo G, Gruhler A, Liu Y, Jensen ON and Dickson RC 2008 The sphingolipid long-chain base-Pkh1/2-Ypk1/2 signaling pathway regulates eisosome assembly and turnover. J. Biol. Chem. 283 10433–10444

    Article  PubMed  CAS  Google Scholar 

  • Malinska K, Malinsky J, Opekarova M and Tanner W 2004 Distribution of Can1p into stable domains reflects lateral protein segregation within the plasma membrane of living S. cerevisiae cells. J. Cell Sci. 117 6031–6041

    Article  PubMed  CAS  Google Scholar 

  • Nannapaneni S, Wang D, Jain S, Schroeder B, Highfill C, Reustle L, Pittsley D, Maysent A, et al. 2010 The yeast dynamin-like protein Vps1:vps1 mutations perturb the internalization and the motility of endocytic vesicles and endosomes via disorganization of the actin cytoskeleton. Eur. J. Cell Biol. 89 499–508

    Article  PubMed  CAS  Google Scholar 

  • Ortiz D and Novick PJ 2006 Ypt32p regulates the translocation of Chs3p from an internal pool to the plasma membrane. Eur. J. Cell Biol. 85 107–116

    Article  PubMed  CAS  Google Scholar 

  • Pruyne D, Gao L, Bi E and Bretscher A 2004 Stable and dynamic axes of polarity use distinct formin isoforms in budding yeast. Mol. Biol. Cell 15 4971–4989

    Article  PubMed  CAS  Google Scholar 

  • Riezman H 1985 Endocytosis in yeast: several of the yeast secretory mutants are defective in endocytosis. Cell 40 1001–1009

    Article  PubMed  CAS  Google Scholar 

  • Schmidt A, Kunz J and Hall MN 1996 TOR2 is required for organization of the actin cytoskeleton in yeast. Proc. Natl. Acad. Sci. USA 93 13780–13785

    Article  PubMed  CAS  Google Scholar 

  • Shaw JD, Cummings KB, Huyer G, Michaelis S and Wendland B 2001 Yeast as a model system for studying endocytosis. Exp. Cell Res. 271 1–9

    Article  PubMed  CAS  Google Scholar 

  • Sizonenko GI, Karpova TS, Gattermeir DJ and Cooper JA 1996 Mutational analysis of capping protein function in Saccharomyces cerevisiae. Mol. Biol. Cell 7 1–15

    PubMed  CAS  Google Scholar 

  • Sweeney HL and Houdusse A 2007 What can myosin VI do in cells? Curr. Opin. Cell Biol. 19 57–66

    Article  PubMed  CAS  Google Scholar 

  • Toshima JY, Toshima J, Kaksonen M, Martin AC, King DS and Drubin DG 2006 Spatial dynamics of receptor-mediated endocytic trafficking in budding yeast revealed by using fluorescent alpha-factor derivatives. Proc. Natl. Acad. Sci. USA 103 5793–5798

    Article  PubMed  CAS  Google Scholar 

  • Vida TA and Emr SD 1995 A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128 779–792

    Article  PubMed  CAS  Google Scholar 

  • Walther TC, Aguilar PS, Frohlich F, Chu F, Moreira K, Burlingame AL and Walter P 2007 Pkh-kinases control eisosome assembly and organization. EMBO J. 26 4946–4955

    Article  PubMed  CAS  Google Scholar 

  • Walther TC, Brickner JH, Aguilar PS, Bernales S, Pantoja C and Walter P 2006 Eisosomes mark static sites of endocytosis. Nature (London) 439 998–1003

    Article  CAS  Google Scholar 

  • Wedlich-Soldner R, Altschuler S, Wu L and Li R 2003 Spontaneous cell polarization through actomyosin-based delivery of the Cdc42 GTPase. Sci. NY 299 1231–1235

    Article  CAS  Google Scholar 

  • Wiederkehr A, Avaro S, Prescianotto-Baschong C, Haguenauer-Tsapis R and Riezman H 2000 The F-box protein Rcy1p is involved in endocytic membrane traffic and recycling out of an early endosome in Saccharomyces cerevisiae. J. Cell Biol. 149 397–410

    Article  PubMed  CAS  Google Scholar 

  • Winter D, Podtelejnikov AV, Mann M and Li R 1997 The complex containing actin-related proteins Arp2 and Arp3 is required for the motility and integrity of yeast actin patches. Curr. Biol. 7 519–529

    Article  PubMed  CAS  Google Scholar 

  • Yu JW, Mendrola JM, Audhya A, Singh S, Keleti D, DeWald DB, Murray D, Emr SD and Lemmon MA 2004 Genome-wide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains. Mol. Cell 13 677–688

    CAS  Google Scholar 

  • Zhang X, Lester RL and Dickson RC 2004 Pil1p and Lsp1p negatively regulate the 3-phosphoinositide-dependent protein kinase-like kinase Pkh1p and downstream signaling pathways Pkc1p and Ypk1p. J. Biol. Chem. 279 22030–22038

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to Scott Emr for providing slm ts mutant strains and Mark Longtine for providing the PCR templates. We thank Paul Durham and Mark Richter for providing unrestricted access to laboratories and equipment. This work was supported by a National Scientific Foundation Grant 0923024 (to KK) and by thesis funding from Missouri State University (to CK).

Author information

Authors and Affiliations

  1. Department of Biology, Missouri State University, 901 South National Ave, Springfield, MO, 65897, USA

    Chitra Kamble, Sandhya Jain, Erin Murphy & Kyoungtae Kim

Authors
  1. Chitra Kamble
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sandhya Jain
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Erin Murphy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kyoungtae Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kyoungtae Kim.

Additional information

[Kamble C, Jain S, Murphy E and Kim K 2011 Requirements of Slm proteins for proper eisosome organization, endocytic trafficking and recycling in the yeast Saccharomyces cerevisiae. J. Biosci. 36 XXX–XXX] DOI

Chitra Kamble and Sandhya Jain have contributed equally to this work.

ePublication: 00 January 2011

Corresponding editor: Durgadas P Kasbekar

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kamble, C., Jain, S., Murphy, E. et al. Requirements of Slm proteins for proper eisosome organization, endocytic trafficking and recycling in the yeast Saccharomyces cerevisiae . J Biosci 36, 79–96 (2011). https://doi.org/10.1007/s12038-011-9018-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12038-011-9018-0

Keywords

  • Actin
  • Actin patch
  • Eisosome
  • Endocytosis
  • Endosome
  • FM4-64
  • Pil1
  • Recycling
  • Slm1/2
  • Trafficking