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Three-dimensional and immune electron microscopic analysis of the secretory pathway in Saccharomyces cerevisiae

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Abstract

Until now, the mechanisms of ER-to-Golgi and intra-Golgi transport remain obscure. This is especially evident for the Golgi of S. cerevisiae where different Golgi compartments are not organized in stacks. Here, using improved sample preparation protocols, we examined the 3D organization of pre-Golgi and Golgi compartments and found several new features of the structures functioning along the secretory pathway. In the cytoplasmic sheet ER, we found narrow pores that aggregated near the rims, and tubular networks tightly interconnected with sheets of several cytoplasmic ER cisternae. Within the Golgi compartments, we found disks with wide pores, disks with narrow pores, and disk-like networks with varicosities or nodules at the point of branching and thick membranes. Sometimes, these compartments contained 30 nm buds coated with a clathrin-like coat. The lumen of these Golgi compartments was more osmiophilic than the lumen of the ER. In contrast to ER elements, Golgi compartments were isolated and in the majority of cases not connected, although we observed some connections between Golgi compartments and also between Golgi disks with wide pores and the ER. Two types of free vesicles of 35–40 and 45–50 nm were found, the former being sometimes partially coated with a clathrin-like coat. Sec31, a COPII component, was found near narrow pores in the cytoplasmic sheets of the ER, over edge aggregates of narrow pores, and within the ER network. The cis-Golgi marker Rer1p was detected on disks or semi-spheres with wide pores, while the medial Golgi marker Gos1p was found on disks or semi-spheres with narrow pores. Gos1p was found to be enriched on 45–50 nm vesicles, while Rer1p was depleted. The 35–40 nm vesicles did not show either label. These findings are discussed from the point of view of mechanisms of transport.

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Acknowledgments

We thank Drs. P. Lupetti (University of Siena, Italy), A. Ellinger, and M. Pavelka (University of Vienna, Austria) for assistance with quick-freezing and high-pressure-freezing experiments; Dr. A. Fusella for the help in EM preparations, and Celeste Pirozzoli for creating the pUG36-Gos1 construct. We acknowledge Italian FIRC and Consorzio Mario Negri Sud for financial support and the Centre European of Nano-medicine (CEN Italy) for the possibility to use the Tecnai 20 electron microscope.

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Authors and Affiliations

  1. Istituto FIRC di Oncologia Molecolare (IFOM-IEO Campus), Via Adamello 16, 20139, Milan, Italy

    Galina V. Beznoussenko & Alexander A. Mironov

  2. Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Via Nazionale 8/A, 66030, Santa Maria Imbaro, CH, Italy

    Galina V. Beznoussenko, Antonella Ragnini-Wilson, Cathal Wilson & Alexander A. Mironov

  3. Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133, Rome, Italy

    Antonella Ragnini-Wilson

  4. Telethon Institute of Genetics and Medicine, Via Campi Flegrei, 34, 80078, Pozzuoli, Naples, Italy

    Cathal Wilson

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  1. Galina V. Beznoussenko
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Fig. S1

Examples of membrane organelles in yeast after application of the OTOTO method with double-step image acquisition. a Mitochondria (arrow). The membranes are well visible. b A full series of virtual sections of 50 nm cytosolic vesicles. c A series of virtual sections taken from the middle part of tomography serial reconstructions. In c10, the dashed white box shows the place where the ER edge network could be situated (see also Fig. 1c). Scale bars: 110 nm panel a; 125 nm panel b (upper row); 100 nm panel b (lower row); 1000 nm panel c. (TIFF 2931 kb)

Fig. S2

Structure of Golgi compartments in yeast cells after freezing and cryo-substitution. Samples were examined using routine electron microscopy (a–e, f–j) or subjected to electron tomography (k, l). a–c Serial sections of the Golgi with an irregular network-like shape (arrow). A few vesicles (dark dots in b) are visible around the Golgi. d, f–j Serial sections of a disk-like Golgi compartment (white arrow in g) containing a COPI-coated bud (arrow in i). Thick arrow in (e) shows medial Golgi compartment, thin arrow in (e) indicates COPI-dependent vesicle. k, l Serial virtual tomography sections of the medial Golgi compartment. The black arrow shows a COPI-coated bud. The white arrow indicates a 47 nm vesicle. Scale bars: 250 nm panels a–c, e; 75 nm panels d, f–l. (TIFF 2931 kb)

Fig. S3

Cryo-sections of cells showing different types of Golgi compartments (arrows): cis (c; thin arrow in f); medial (d; white arrow in f); medial with a spheroid shaped (e), trans (thick arrow in f); aggregation of pores near the ER rim (g); secretory granules (h). Scale bars: 500 nm panels a, b; 75 nm panels c–e, h; 120 nm panels f, g. (TIFF 2931 kb)

Fig. S4

Cells expressing GFP-Gos1p, a medial Golgi marker, after labeling with an antibody against GFP (10 nm gold particles). Labeling of 45–50 nm vesicles is visible in (f) and (i). Scale bar, 150 nm. (TIFF 2931 kb)

Fig. S5

Three-dimensional view of the elements of the secretory pathway. a The ER edge network shown in Fig. 1 e, f. The ER is colored in green. b Different types of vesicles: blue—secretory granules; red—a 35–40 nm vesicle; orange—45–50 nm vesicles. The ER is colored in dark green and the medial Golgi in light green. Scale bar, 140 nm. (TIFF 3598 kb)

Fig. S6

Identification of Golgi compartments using CLEM. Yeast cells expressing GFP-Rer1p were plated on concanavalin-coated MatTek gridded plates, examined by fluorescent microscopy under living conditions and then fixed (see Materials and methods). a, b Bright field (low and high magnification, respectively) of the fluorescent images of cells shown in (c) was acquired. The samples were then processed for EM. d Initial sections of the cells indicated by white arrow in (a-c). e, f Golgi structures corresponding to the indicated fluorescent spots and visible in different serial sections. The cis-Golgi contained a lot of perforations. Scale bars: 30 µm panel a; 3 µm panel b; 2 µm panels c, d; 500 nm panels e, f. (TIFF 2931 kb)

Table. S1

Labeling density of structures after immune-EM with the indicated probe (DOCX 10 kb)

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Beznoussenko, G.V., Ragnini-Wilson, A., Wilson, C. et al. Three-dimensional and immune electron microscopic analysis of the secretory pathway in Saccharomyces cerevisiae . Histochem Cell Biol 146, 515–527 (2016). https://doi.org/10.1007/s00418-016-1483-y

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