Letter | Published:

Golgi maturation visualized in living yeast

Naturevolume 441pages10021006 (2006) | Download Citation

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Abstract

The Golgi apparatus is composed of biochemically distinct early (cis, medial) and late (trans, TGN) cisternae. There is debate about the nature of these cisternae1,2,3. The stable compartments model predicts that each cisterna is a long-lived structure that retains a characteristic set of Golgi-resident proteins. In this view, secretory cargo proteins are transported by vesicles from one cisterna to the next. The cisternal maturation model predicts that each cisterna is a transient structure that matures from early to late by acquiring and then losing specific Golgi-resident proteins. In this view, secretory cargo proteins traverse the Golgi by remaining within the maturing cisternae. Various observations have been interpreted as supporting one or the other mechanism4,5,6,7,8,9. Here we provide a direct test of the two models using three-dimensional time-lapse fluorescence microscopy of the yeast Saccharomyces cerevisiae. This approach reveals that individual cisternae mature, and do so at a consistent rate. In parallel, we used pulse–chase analysis to measure the transport of two secretory cargo proteins. The rate of cisternal maturation matches the rate of protein transport through the secretory pathway, suggesting that cisternal maturation can account for the kinetics of secretory traffic.

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Change history

  • 22 June 2006

    In the AOP version (PDF only) of this Article, the rightmost edge of Fig. 3b was cut off. The PDF and print versions have been corrected.

References

  1. 1

    Glick, B. S. & Malhotra, V. The curious status of the Golgi apparatus. Cell 95, 883–889 (1998)

  2. 2

    Pelham, H. R. & Rothman, J. E. The debate about transport in the Golgi—two sides of the same coin? Cell 102, 713–719 (2000)

  3. 3

    Rabouille, C. & Klumperman, J. Opinion: The maturing role of COPI vesicles in intra-Golgi transport. Nature Rev. Mol. Cell Biol. 6, 812–817 (2005)

  4. 4

    Bonfanti, L. et al. Procollagen traverses the Golgi stack without leaving the lumen of cisternae: evidence for cisternal maturation. Cell 95, 993–1003 (1998)

  5. 5

    Wooding, S. & Pelham, H. R. B. The dynamics of Golgi protein traffic visualized in living yeast cells. Mol. Biol. Cell 9, 2667–2680 (1998)

  6. 6

    Volchuk, A. et al. Megavesicles implicated in the rapid transport of intracisternal aggregates across the Golgi stack. Cell 102, 335–348 (2000)

  7. 7

    Mironov, A. A. et al. Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae. J. Cell Biol. 155, 1225–1238 (2001)

  8. 8

    Martínez-Menàrguez, J. A. et al. Peri-Golgi vesicles contain retrograde but not anterograde proteins consistent with the cisternal progression model of intra-Golgi transport. J. Cell Biol. 155, 1213–1224 (2001)

  9. 9

    Cosson, P., Amherdt, M., Rothman, J. E. & Orci, L. A resident Golgi protein is excluded from peri-Golgi vesicles in NRK cells. Proc. Natl Acad. Sci. USA 99, 12831–12834 (2002)

  10. 10

    Franzusoff, A., Redding, K., Crosby, J., Fuller, R. S. & Schekman, R. Localization of components involved in protein transport and processing through the yeast Golgi apparatus. J. Cell Biol. 112, 27–37 (1991)

  11. 11

    Preuss, D., Mulholland, J., Franzusoff, A., Segev, N. & Botstein, D. Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy. Mol. Biol. Cell 3, 789–803 (1992)

  12. 12

    Payne, W. E., Gannon, P. M. & Kaiser, C. A. An inducible acid phosphatase from the yeast Pichia pastoris: characterization of the gene and its product. Gene 163, 19–26 (1995)

  13. 13

    Mogelsvang, S., Gomez-Ospina, N., Soderholm, J., Glick, B. S. & Staehelin, L. A. Tomographic evidence for continuous turnover of Golgi cisternae in Pichia pastoris. Mol. Biol. Cell 14, 2277–2291 (2003)

  14. 14

    Duden, R. & Schekman, R. in The Golgi Apparatus (eds Berger, E. G. & Roth, J.) 219–246 (Birkhäuser Verlag, Basel, 1997)

  15. 15

    Bevis, B. J., Hammond, A. T., Reinke, C. A. & Glick, B. S. De novo formation of transitional ER sites and Golgi structures in Pichia pastoris. Nature Cell Biol. 4, 750–756 (2002)

  16. 16

    Hammond, A. T. & Glick, B. S. Raising the speed limits for 4D fluorescence microscopy. Traffic 1, 935–940 (2000)

  17. 17

    Rossanese, O. W. et al. A role for actin, Cdc1p and Myo2p in the inheritance of late Golgi elements in Saccharomyces cerevisiae. J. Cell Biol. 153, 47–61 (2001)

  18. 18

    Dean, N., Zhang, Y. B. & Poster, J. B. The VRG4 gene is required for GFP-mannose transport into the lumen of the golgi in the yeast, Saccharomyces cerevisiae. J. Biol. Chem. 272, 31908–31914 (1997)

  19. 19

    Abe, M., Noda, Y., Adachi, H. & Yoda, K. Localization of GDP-mannose transporter in the Golgi requires retrieval to the endoplasmic reticulum depending on its cytoplasmic tail and coatomer. J. Cell Sci. 117, 5687–5696 (2004)

  20. 20

    Behnia, R., Panic, B., Whyte, J. R. & Munro, S. Targeting of the Arf-like GTPase Arl3p to the Golgi requires N-terminal acetylation and the membrane protein Sys1p. Nature Cell Biol. 6, 405–413 (2004)

  21. 21

    McNew, J. A. et al. Gos1p, a Saccharomyces cerevisiae SNARE protein involved in Golgi transport. FEBS Lett. 435, 89–95 (1998)

  22. 22

    Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003)

  23. 23

    Brigance, W. T., Barlowe, C. & Graham, T. R. Organization of the yeast Golgi complex into at least four functionally distinct compartments. Mol. Biol. Cell 11, 171–182 (2000)

  24. 24

    Horazdovsky, B. F., DeWald, D. B. & Emr, S. D. Protein transport to the yeast vacuole. Curr. Opin. Cell Biol. 7, 544–551 (1995)

  25. 25

    Matsuura-Tokita, K., Takeuchi, M., Ichihara, A., Mikuriya, K. & Nakano, A. Live imaging of yeast Golgi cisternal maturation. Nature advance online publication, doi:10.1038/nature04737 (14 May 2006)

  26. 26

    Morin-Ganet, M.-N., Rambourg, A., Deitz, S. B., Franzusoff, A. & Képès, F. Morphogenesis and dynamics of the yeast Golgi apparatus. Traffic 1, 56–68 (2000)

  27. 27

    Todorow, Z., Spang, A., Carmack, E., Yates, J. & Schekman, R. Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum. Proc. Natl Acad. Sci. USA 97, 13643–13648 (2000)

  28. 28

    Reinke, C. A., Kozik, P. & Glick, B. S. Golgi inheritance in Saccharomyces cerevisiae depends on ER inheritance. Proc. Natl Acad. Sci. USA 101, 18018–18023 (2004)

  29. 29

    Trucco, A. et al. Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments. Nature Cell Biol. 6, 1071–1081 (2004)

  30. 30

    Marsh, B. J., Volkmann, N., McIntosh, J. R. & Howell, K. E. Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells. Proc. Natl Acad. Sci. USA 101, 5565–5570 (2004)

  31. 31

    Graham, T. R. in Current Protocols in Cell Biology (ed. Morgan, K.) 7.6.1–7.6.9 (Wiley, New York, 2000)

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Acknowledgements

Thanks to A. Franzusoff, T. Stevens and P. Silver for providing reagents, to A. Hammond for advice about microscopy, and to T. Graham for help with pulse–chase analysis. We are grateful to A. Nakano for discussions and for sharing data before publication. This work was supported by grants from the March of Dimes Birth Defects Foundation, the National Institutes of Health and the American Cancer Society.

Author information

Affiliations

  1. Department of Molecular Genetics and Cell Biology, and Institute for Biophysical Dynamics, The University of Chicago, 920 East 58th Street, Illinois, 60637, Chicago, USA

    • Eugene Losev
    • , Catherine A. Reinke
    • , Jennifer Jellen
    • , Daniel E. Strongin
    • , Brooke J. Bevis
    •  & Benjamin S. Glick

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Competing interests

Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Corresponding author

Correspondence to Benjamin S. Glick.

Supplementary information

  1. Supplementary Figure 1

    Experimental strategy for distinguishing between the cisternal maturation and stable compartments models. (PDF 287 kb)

  2. Supplementary Figure 2a

    Analysis method for the 4D datasets. a, Schematic diagram of the 4D analysis. (JPG 252 kb)

  3. Supplementary Figure 2b

    Analysis method for the 4D datasets. b, Representative z stack of optical sections from Movie 1a, showing cisternae labeled with Sec7p–GFP. (JPG 344 kb)

  4. Supplementary Figure 2c

    Analysis method for the 4D datasets. C, Representative z¬ stack of optical sections from Movie 3a, showing cisternae labeled with GFP–Vrg4p and Sec7p–DsRed. (JPG 817 kb)

  5. Supplementary Figure 3

    Overexpression of Sec7p-DsRed does not change the relative localizations of GFP Vrg4p and Sec7p. (JPG 194 kb)

  6. Supplementary Figure 4

    Strains containing tagged Vrg4p and/or tagged Sec7p exhibit normal trafficking of CPY. (JPG 140 kb)

  7. Supplementary Figure 5

    The labeling of cisternae with GFP–Vrg4p is similar in duration to the labeling with Sec7p–GFP. (JPG 250 kb)

  8. Supplementary Figure 6

    SDS–PAGE and autoradiography data for the pulse-chase analyses of ? factor and carboxypeptidase Y. (JPG 364 kb)

  9. Supplementary Movie 1a

    Sec7p–GFP labelling. (MOV 20470 kb)

  10. Supplementary Movie 1b

    Sec7p–GFP labelling, edited. (MOV 16420 kb)

  11. Supplementary Movie 2a

    Sys1p-GFP and Sec7p–DsRed dual labelling. (MOV 9524 kb)

  12. Supplementary Movie 2b

    Sys1p–GFP and Sec7p–DsRed dual labelling, edited. (MOV 7646 kb)

  13. Supplementary Movie 3a

    GFP-Vrg4p and Sec7p–DsRed dual labelling. (MOV 14696 kb)

  14. Supplementary Movie 3b

    GFP–Vrg4p and Sec7p–DsRed dual labelling, edited. (MOV 9471 kb)

  15. Supplementary Movie S1a

    Animation of a cisternal maturation mechanism for Golgi transport. (MOV 2130 kb)

  16. Supplementary Movie S1b

    Animation of a stable compartments mechanism for Golgi transport. (MOV 423 kb)

  17. Supplementary Movie S2

    Example of a cisterna being tracked in a 4D dataset. (MOV 13644 kb)

  18. Supplementary Movie S3a

    GFP–Vrg4p labelling. (MOV 15081 kb)

  19. Supplementary Movie S3b

    GFP–Vrg4p labelling, edited. (MOV 11665 kb)

  20. Supplementary Table S1

    Duration of labelling of cisternae with Sec7p–GFP or GFP–Vrg4p. (PDF 33 kb)

  21. Supplementary Table S2

    Early Golgi cisternae consistently become late Golgi cisternae. (PDF 26 kb)

  22. Supplementary Notes

    This file contains Supplementary Methods, Supplementary Figure Legends and Supplementary Movie Legends. (DOC 75 kb)

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https://doi.org/10.1038/nature04717

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