A role for actin, Cdc1p, and Myo2p in the inheritance of late Golgi elements in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, Golgi elements are present in the bud very early in the cell cycle. We have analyzed this Golgi inheritance process using fluorescence microscopy and genetics. In rapidly growing cells, late Golgi elements show an actin-dependent concentration at sites of polarized growth. Late Golgi elements are apparently transported into the bud along actin cables and are also retained in the bud by a mechanism that may involve actin. A visual screen for mutants defective in the inheritance of late Golgi elements yielded multiple alleles of CDC1. Mutations in CDC1 severely depolarize the actin cytoskeleton, and these mutations prevent late Golgi elements from being retained in the bud. The efficient localization of late Golgi elements to the bud requires the type V myosin Myo2p, further suggesting that actin plays a role in Golgi inheritance. Surprisingly, early and late Golgi elements are inherited by different pathways, with early Golgi elements localizing to the bud in a Cdc1p- and Myo2p-independent manner. We propose that early Golgi elements arise from ER membranes that are present in the bud. These two pathways of Golgi inheritance in S. cerevisiae resemble Golgi inheritance pathways in vertebrate cells.

Figure 1

Sec7p–GFP is concentrated at sites of polarized growth in rapidly growing or schmooing cells. (A) Visualization of actin and Sec7p–GFP through the cell cycle. An unsynchronized culture of BGY316 cells growing rapidly at 30°C was fixed and stained with Alexa 594–phalloidin. The cells were then imaged by DIC microscopy and dual-color fluorescence microscopy. Numbers mark examples of a nonpolarized G1 cell (1), a polarized G1 cell (2), an S phase cell (3), and a cell undergoing cytokinesis (4). Actin patches and Sec7p–GFP are both concentrated at sites of polarized growth. (B) Distributions of actin and Sec7p–GFP in schmooing cells. A BGY316 culture was treated with α-factor for 90 min at 30°C, and then fixed and viewed as in A. Actin patches and Sec7p–GFP are concentrated in the schmoo. (C) Effects of disrupting actin or microtubules on the Sec7p–GFP pattern in rapidly growing cells. Aliquots of a rapidly growing BGY316 culture were either left untreated (DMSO), or treated with 200 μM LatA or 15 μg/ml nocodazole (Noc) for 30 min at 30°C before fixation. A portion of each aliquot was stained with Alexa 594–phalloidin to confirm that actin polymers had been disrupted by LatA, and a second portion was processed for immunofluorescence to confirm that microtubules had been disrupted by nocodazole (not shown). 50 budded cells from each aliquot were examined as in A, and the results from two separate experiments were combined. A cell was scored as having polarized Sec7p–GFP if it displayed a clear concentration of fluorescence in or near the bud. As judged by this assay, the polarization of Sec7p–GFP is effectively abolished by LatA treatment but is unaffected by nocodazole treatment. Bars, 2 μm.

Figure 2

A subset of the late Golgi elements localize to the bud even after LatA treatment. (A and B) As described in the legend to Fig. 1 C, a rapidly growing BGY316 culture was treated with LatA for 30 min before fixation. Merged DIC and fluorescence images of representative LatA-treated cells are shown in A. Sec7p–GFP is generally depolarized, yet most of the small buds contain at least one spot of Sec7p–GFP fluorescence. This residual Sec7p–GFP inheritance is quantified in B. As described in Materials and Methods, budded cells from cultures mock treated with DMSO (○) or treated with LatA (•) were assigned to five categories on the basis of bud size, with category I cells having the smallest buds and category V the largest. Each budded cell was scored in a plus–minus manner for the presence of Sec7p–GFP in the bud. The results indicate that LatA treatment causes only a minor reduction in the percentage of buds containing Sec7p–GFP. (C and D) Cultures carrying the wild-type ACT1 gene (strain BGY316; ○) or the act1-ΔDSE allele (strain BGY414; •) were grown at 30°C, labeled with FM4-64, and fixed. Using the plus–minus visual assay, budded cells were assessed for the inheritance of vacuoles (C) and Sec7p–GFP (D). The act1-ΔDSE mutation inhibits vacuolar inheritance but has no effect on Sec7p–GFP inheritance. Bar, 2 μm.

Figure 5

The small buds of cdc1-304 cells repeatedly lose and regain late Golgi elements. Shown are merged fluorescence and transmitted light projections of a representative budded cdc1-304 cell, which had been incubated at 37°C for 2 h when imaging was initiated. These selected frames from the time series demonstrate that the bud alternately contains and lacks detectable Sec7p–GFP fluorescence. Bar, 2 μm.

Figure 3

Mutants with altered inheritance or morphology of Sec7p–GFP-labeled Golgi elements. Cultures were grown overnight at room temperature and then shifted to 37°C for 1 h before fixation. (A) Golgi distribution and morphology in the four classes of mutants. DIC and fluorescence images were captured separately and merged. In class A mutants, the Sec7p–GFP-labeled Golgi elements exhibit normal size and distribution within the mother cell, but are frequently absent from the small buds. Strain BGY105 is shown. Class B mutants contain larger, fewer Golgi elements than wild-type cells. A typical class B cell has one to three large spots of Sec7p–GFP fluorescence. In class C mutants, the Golgi elements are smaller and more numerous than in wild-type cells. Moreover, class C mutants often display an even stronger polarization of late Golgi elements than do wild-type cells. The single class D mutant has a variety of Golgi morphology defects: Sec7p–GFP fluorescence can appear as rings, bars, or thin lines. (B) Cultures of wild-type cells (strain BGY316; ○) and a representative class A mutant (strain BGY111; •) were grown at room temperature and then shifted to 37°C for 1 h before fixation. Quantitation by the plus–minus visual assay confirmed that Sec7p–GFP inheritance is defective in the class A mutant. Bar, 2 μm.

Figure 4

The cdc1-304 mutant exhibits defects in actin polarity and vacuolar inheritance. (A) Wild-type (BGY316) and cdc1-304 (BGY111) cultures were grown at room temperature, shifted to 37°C for 1 h, fixed, and stained with Alexa 594–phalloidin. Fluorescence images are shown. Most of the wild-type cells contain polarized actin patches and cables, whereas most of the cdc1-304 cells contain depolarized patches and few visible cables. (B) Wild-type and cdc1-304 cultures were grown at room temperature, labeled with FM4-64, and then shifted to 37°C for 1 h. The presence of maternally derived vacuolar material in the bud was quantified as described in the legend to Fig. 2 C. The cdc1-304 mutant shows a delay in vacuolar inheritance. Bar, 2 μm.

Figure 8

Creation of two additional GFP-labeled Golgi markers. (A) Representative cells expressing SEC21-GFPx3 or GFP-YPT1. Shown are merged DIC and fluorescence images. (B) Double label immunofluorescence of Sec7p with either Sec21p–GFP or GFP–Ypt1p. Fixed cells were labeled with polyclonal anti-Sec7p antibody followed by Rhodamine red-X–conjugated anti–rabbit antibody (red) and with monoclonal anti-GFP antibody followed by Cy2-conjugated anti–mouse antibody (green). As shown in the merged images, Sec7p shows partial colocalization with Sec21p–GFP but no significant colocalization with GFP–Ypt1p. Bars, 2 μm.

Figure 6

Defective inheritance of vacuoles and Sec7p–GFP in myo2-66 cells. Cultures carrying the wild-type MYO2 gene (strain BGY316; ○) or the myo2-66 allele (strain BGY415; •) were grown at 23°C and assayed as in Fig. 2 for the inheritance of vacuoles (A) or Sec7p–GFP (B).

Figure 7

Creation of a second late Golgi marker. (A) Representative cells expressing RIC1–GFPx3. Shown is a merged DIC and fluorescence image. (B) Inheritance of Ric1p–GFP in wild-type and mutant cells. As described in the legend to Fig. 3 B, a wild-type strain (○), a cdc1-304 strain (•), and a myo2-66 strain (□) were assayed for the inheritance of Ric1p–GFP. The wild-type and cdc1-304 strains were grown at 23°C and then shifted to 37°C for 1 h before fixation. The myo2-66 strain was maintained at 23°C before fixation, but the myo2-66 data can be compared with the wild-type data because Golgi inheritance in wild-type cells is unaffected by shifting to 37°C for 1 h (not shown). The inheritance of Ric1p–GFP is significantly inhibited by the cdc1-304 and myo2-66 mutations. Bar, 2 μm.

Figure 10

The ER and tER are inherited normally in cdc1-304 cells. (A) Fluorescence images of wild-type (BGY418) and cdc1-304 (BGY419) strains expressing GFP–HDEL. This protein marks the nuclear envelope as well as peripheral ER membranes. Cells were grown at 23°C and then shifted to 37°C for 1 h before fixation. (B) Merged DIC and fluorescence images of wild-type (BGY318) and cdc1-304 (BGY110) strains expressing SEC13-GFP. This fusion protein marks the nascent COPII vesicles that define the tER. Both the ER and the tER localize to small buds with undiminished efficiency in the cdc1-304 mutant. Bars, 2 μm.

Figure 9

Inheritance of early Golgi markers in wild-type and mutant cells. As described in the legend to Fig. 3 B, wild-type strains (○), cdc1-304 strains (•), and myo2-66 strains (□) were assayed for the inheritance of Sec21p–GFP (A) and GFP–Ypt1p (B). The wild-type and cdc1-304 strains were grown at 23°C and then shifted to 37°C for 1 h before fixation, whereas the myo2-66 strains were maintained at 23°C before fixation (see Fig. 7). The inheritance of Sec21p–GFP is unaffected by the cdc1-304 mutation and is inhibited only slightly by the myo2-66 mutation, whereas the inheritance of GFP–Ypt1p is not affected by either mutation.

Figure 11

Model for Golgi inheritance in S. cerevisiae. (A) Inheritance of early Golgi elements. The ER comprises the nuclear envelope plus peripheral ER membranes. COPII vesicles bud throughout the ER network, thereby generating early Golgi cisternae in both the mother cell and the bud. The inheritance of early Golgi elements requires ER inheritance but does not directly require actin cables, Myo2p or Cdc1p. (B) Inheritance of late Golgi elements. Dimeric Myo2p molecules bind to late Golgi cisternae and transport these cisternae along actin cables into the bud. Additional Myo2p molecules retain a subset of the late Golgi cisternae in the bud by binding to actin or other bud components. Cdc1p is required to maintain a polarized actin cytoskeleton and to retain late Golgi cisternae in the bud.