19S and 20S proteasome subunits colocalization. (A) Colocalization of 19S RP and 20S CP proteasome subunits in quiescent yeast cells. Cells coexpressing the indicated fusion proteins were imaged after 4 d of growth in YPDA at 30°C. GFP fluorescence is green and RFP fluorescence is red. Images are single focal planes. (B) Cells expressing Scl1p-GFP and Pup1p-RFP were grown in YPDA medium at 30°C. For each time point, cells displaying both the green and the red fluorescence were scored as indicated in the Fig. 1 legend. For each time point, n > 200 (two experiments; error bars show SD). Typical colocalization images corresponding to each type of proteasome localization pattern are shown on the right. Images are maximal projection of z stacks. Bars, 2 μm.

PSGs are rapidly mobilized upon exit from quiescence. Cells expressing Pre6p-GFP were grown for 4 d in YPD medium at 30°C, washed, and either resuspended in water (A) or new YPD medium (B) or preincubated for 1 h in old YPD medium containing 100 μg/ml cycloheximide and then released into new YPD medium containing 100 μg/ml cycloheximide (C). For each time point, cells displaying Pre6p-GFP fluorescence within PSGs, immobile dots close to the nuclear periphery, nuclear periphery, or nucleus were counted (n > 200; two experiments; error bars show SD). The budding index is indicated (n > 200; two experiments; SD < 5%). Images are maximal projection of z stacks. Bar, 2 μm. Of note, the levels of gray for images where PSGs are detected range from 0 to 1,500, and they range from 0 to 500 for images where the fluorescence is detected at the nuclear periphery or in the nucleus.

PSGs, actin bodies, and P-bodies are distinct structures. (A) PSGs, as revealed by Pre6p-GFP fluorescence, were observed in cells grown for 4 d in different carbon sources containing rich media and in diploid cells grown in YPDA. For each condition n > 200 (two experiments; error bars show SD). Typical images (maximal projection of z stacks) of cells displaying PSGs (Pre6p-GFP) are shown on the right. (B) Cells expressing Pre6p-GFP were grown in YPDA medium at 30°C, fixed, and stained with Alexa Fluor phalloidin to reveal F-actin–containing structures. Actively proliferating cells (left) displayed actin patches and cables (red) and a typical Pre6p-GFP nuclear localization. Quiescent cells displayed PSGs (green fluorescence) that did not colocalize with actin bodies (red). (C) Cells coexpressing Dcp2p-GFP and Pup1p-RFP were grown in YPDA medium at 30°C. Actively proliferating yeast cells displayed a typical Pup1p-RFP nuclear localization and small and discrete Dcp2p-GFP dots. In quiescent cells, Dcp2p-GFP localized in P-bodies that did not colocalize with PSGs. Images in B and C are single focal planes. Bars, 2 μm.

S. pombe cells display PSG.S. pombe expressing Pad1-GFP (Rpn11 homologue) were grown for 4 d in YPDA medium at 30°C, washed, and resuspended either in water (A) or new YPDA medium (B) or preincubated for 1 h in old YPDA medium containing 100 μg/ml cycloheximide and then released into new YPDA medium containing 100 μg/ml cycloheximide (C). For each time point, cells displaying Pad1-GFP fluorescence within PSG or at the nuclear periphery were counted (n > 200; two experiments; error bars show SD). Images are maximal projection of z stacks. Bar, 2 μm. Of note, cycloheximide was active on S. pombe cells because 2 h after refeeding in the presence of the drug, cells did not grow and displayed a round-shape morphology, whereas the untreated cells elongated and started to divide. The mean cell size before exit from quiescence was 7.44 ± 0.86 μm (n = 50). 3 h after transfer of quiescent cells to new YPDA medium, mean cell size increased (13 ± 2 μm; n = 50). In contrast, 3 h after transfer of quiescent cells to new YPDA medium containing 100 μg/ml cycloheximide the cell size did not significantly change (8.35 ± 1.16 μm; n = 50).

Proteasome relocalization upon entry into quiescence. (A–C) Cells expressing Pre6p-GFP were grown in YPDA medium at 30°C. (A) At various stages of growth, OD 600 nm was monitored (red curve), glucose concentration in the medium was measured (green curve), and the budding index was determined (blue curve; n > 200 cells for each time point). (B) Localization of Pre6p-GFP. For each time point, the localization of Pre6p-GFP fluorescence was scored as nuclear (green bar), at the nuclear periphery (blue bar), immobile dots close to the nuclear periphery (yellow bar), or bright mobile cytoplasmic dots (PSG) with or without nuclear periphery staining (red bar; n > 200 cells for each time point; two experiments; error bars show SD). The gray bars represent the percentage of cells in which GFP fluorescence was not detectable. (C) Typical images of each Pre6p-GFP localization patterns are shown. Images are maximal projection of 0.2-μm z stacks. Of note, for localization of Pre6p-GFP at the nuclear periphery, images were taken with an exposure time that was three times longer than for the other images. Furthermore, levels of gray of the top two rows of images range from 0 to 800 and the levels of gray of the two bottom rows of images range from 0 to 3,000. (D) Colocalization of the proteasome (as revealed by the 20S CP subunit Pup1p fused to RFP) and the nuclear envelope (as revealed by the nuclear pore complex protein Nup2p fused to GFP). (E) Colocalization of the proteasome (as revealed by the 20S CP subunit Pup1p fused to RFP) and the endoplasmic reticulum (as revealed by the membrane protein Pho88p fused to GFP). Images in D and E are single focal planes. Bars, 2 μm. (F) Immunolocalization using anti-20S CP antibodies detected with secondary antibodies linked to 10-nm gold particles in wild-type yeast cells grown for 4 d at 30°C in YPDA medium.