Golgi maturation visualized in living yeast

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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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Figure 1: Sec7–GFP labels each Golgi cisterna for approximately 2 min.
Figure 2: Two markers of the late Golgi show very similar dynamics.
Figure 3: The resident Golgi protein composition of each cisterna changes over time.
Figure 4: Secretory pathway transport kinetics are consistent with cisternal maturation.

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.

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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.

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Correspondence to Benjamin S. Glick.

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Supplementary information

Supplementary Figure 1

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

Supplementary Figure 2a

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

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)

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)

Supplementary Figure 3

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

Supplementary Figure 4

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

Supplementary Figure 5

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

Supplementary Figure 6

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

Supplementary Movie 1a

Sec7p–GFP labelling. (MOV 20470 kb)

Supplementary Movie 1b

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

Supplementary Movie 2a

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

Supplementary Movie 2b

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

Supplementary Movie 3a

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

Supplementary Movie 3b

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

Supplementary Movie S1a

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

Supplementary Movie S1b

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

Supplementary Movie S2

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

Supplementary Movie S3a

GFP–Vrg4p labelling. (MOV 15081 kb)

Supplementary Movie S3b

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

Supplementary Table S1

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

Supplementary Table S2

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

Supplementary Notes

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

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Losev, E., Reinke, C., Jellen, J. et al. Golgi maturation visualized in living yeast. Nature 441, 1002–1006 (2006). https://doi.org/10.1038/nature04717

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