The dynamics of golgi protein traffic visualized in living yeast cells

Abstract

We describe for the first time the visualization of Golgi membranes in living yeast cells, using green fluorescent protein (GFP) chimeras. Late and early Golgi markers are present in distinct sets of scattered, moving cisternae. The immediate effects of temperature-sensitive mutations on the distribution of these markers give clues to the transport processes occurring. We show that the late Golgi marker GFP-Sft2p and the glycosyltransferases, Anp1p and Mnn1p, disperse into vesicle-like structures within minutes of a temperature shift in sec18, sft1, and sed5 cells, but not in sec14 cells. This is consistent with retrograde vesicular traffic, mediated by the vesicle SNARE Sft1p, to early cisternae containing the target SNARE Sed5p. Strikingly, Sed5p itself moves rapidly to the endoplasmic reticulum (ER) in sec12 cells, implying that it cycles through the ER. Electron microscopy shows that Golgi membranes vesiculate in sec18 cells within 10 min of a temperature shift. These results emphasize the dynamic nature of Golgi cisternae and satisfy the kinetic requirements of a cisternal maturation model in which all resident proteins must undergo retrograde vesicular transport, either within the Golgi complex or from there to the ER, as anterograde cargo advances.

Figure 1

Localization of GFP-Sed5p and GFP-Sft2p. (A) Comparison of GFP fluorescence with immunofluorescent staining of marker proteins. Each pair of panels shows the same cells with the indicated markers; arrows indicate similarities and differences. The comparisons with Anp1p are projections of multiple confocal images spanning the entire thickness of the cells; others are single confocal planes. (B) Immunoblots of whole-cell extracts from strains expressing the indicated chimeras, probed with anti-GFP or anti-Sed5p. (C) Double label with two different GFP variants. A reconstruction of a single living cell is shown, viewed from above (top) and from the side (bottom). Streaking in the side view is due to lower optical resolution in the vertical dimension. Red corresponds to GFP-Sed5p; green represents an Sft2p chimera with the W7 variant of GFP.

Figure 2

Movement of Golgi elements. Panel A shows two images of live cells expressing GFP-Sft2p, taken 1-min apart. Four optical sections spaced 1.5 μm apart (spanning the cell thickness) have been projected into a single image to minimize changes caused merely by objects shifting out of one focal plane. Arrows indicate differences in some, but not all, structures. Panel B shows single optical sections of a cell expressing GFP-Sed5p, taken 1.15 s apart. Arrows indicate the original and new position of one structure in this plane, which appears to undergo rapid movement. Bar, 1 μm.

Figure 3

Dispersal of GFP-Sft2p in a sec18 mutant. The upper panels show a control with SEC⁺ cells, the same group of cells being imaged at 20°C, 10 min after initiation of warming to 37°C (i.e., after about 8 min at this temperature), and after re-equilibration to 20°C (20 min after heating stopped). The lower panels show the equivalent experiment with sec18 cells. In the 37°C panel one cell (outlined in white) is printed with the brightness increased, to show more clearly the lack of punctate staining under these conditions. Images are projections of four optical sections. The graphs show smoothed profiles of the distribution of pixel brightness in groups of five to eight cells imaged successively at low and high temperature, obtained as described in MATERIALS AND METHODS. Note the loss of the brighter pixels at 37°C in the sec18 cells.

Figure 4

Dispersal of GFP-Sft2p in sft1 and sed5 mutants. Experiments were performed as in Figure 3. The dark areas in the cells at 37°C correspond to vacuoles. Note the loss and subsequent recovery of bright pixels revealed by the graphs. The slight residual punctate pattern in sft1–15 cells may be due to incomplete inactivation of Sft1p; other alleles showed less of this but also had a partial phenotype at 20°C.

Figure 5

Specificity of the dispersal phenomenon. Experiments were performed as in Figures 3 and 4. (A) unlike GFP-Sft2p, GFP-Sed5p does not disperse in sft1 cells. (B) GFP-Sft2p does not disperse in sec14 cells. In this experiment the 37° image was taken 16 min after initiation of warming to ensure full onset of the secretory block; the pattern did not change during a further 20-min incubation at 37°C.

Figure 6

GFP-Sed5p moves to the ER in sec12 cells. (A) Single optical sections showing GFP-Sed5p in sec12 cells at 20°C and at 37°C (30 min after initiation of warming). Arrows indicate nuclear envelope fluorescence (compare BiP staining in Figure 1). (B) Control showing GFP-Sed5p in SEC⁺ cells. (C) Time course of movement of GFP-Sed5p in sec12 cells. Images (single optical sections) were taken at the times indicated (min), zero being the point at which heating was initiated. Just before the −1 time point, the left half of the field was photobleached. Note that the loss of punctate fluorescence and the emergence of the ER pattern in the unbleached cells occurred more rapidly than the reappearance of fluorescence in the bleached cells.

Figure 7

GFP-Sft2p dispersal in sec12 cells. The experiment was performed as in Figures 3 and 4.

Figure 8

Dispersal of the early-Golgi marker Anp1p. (A) Cells of the indicated genotypes expressing epitope-tagged Sed5p were shifted from 20°C to 37°C for 15 min, fixed, and stained with appropriate antibodies to detect the tagged Sed5p. Arrows indicate nuclear envelope (i.e., ER) staining. (B) Cells of the indicated genotypes were incubated at 20°C or shifted to 37°C for 15 min, fixed, and stained with anti-Anp1p antibodies. Single confocal sections are shown in each case.

Figure 9

Subcellular fractionation of Golgi proteins in sec12 cells. SEC⁺ and sec12 strains expressing either GFP-Sft2p or a myc-tagged version of Emp47p (Lewis and Pelham, 1996) were incubated at 37°C for 90 min, and medium-speed (p13) and high-speed (p100) pellet fractions were prepared. These were immunoblotted with appropriate antibodies as indicated.

Figure 10

Dispersal of the late-Golgi marker Mnn1p. Cells expressing epitope-tagged Mnn1p were incubated at 20°C or for 15 min at 37°C as indicated, fixed, and stained with tag-specific antibodies as in Figure 8. Single confocal sections are shown; the outlined regions have the brightness increased to show the appearance of the dispersed staining more clearly.

Figure 11

Electron microscopy of sec18 cells. Panel A shows cells incubated at 17°C (permissive temperature). Arrows indicate membrane profiles typical of the Golgi apparatus. Panel B shows cells fixed 10 min after a shift to 37°C. Putative Golgi profiles are virtually absent, and many small vesicles have accumulated. Bar, 1 μm.

Figure 12

Model and summary of results. A model of membrane traffic in the yeast Golgi, based on previous results, is depicted. Curved arrows indicate vesicular transport steps, and the steady-state locations of Sft2p, Sft1p, and Sed5p are shown. The sites at which the various mutations act are also shown. Note that only the molecules and effects relevant to the data in this paper are indicated, and that sec18, for example, will block other steps also. Gray arrows represent maturation of cisternae, but the data in this paper do not exclude the possibility of vesicular transport from early to late Golgi. The model accounts for the observations that Sft2p (and Mnn1p, which has a similar location) disperses in small structures (vesicles) in sft1, sed5, sec18, and sec12, but not in sec14, and that Sed5p moves to the ER in sec12 but remains in Golgi cisternae in sft1. Anp1p, although largely colocalizing with Sed5p, recycles within the Golgi rather than returning to the ER.