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Plasma Membrane Compartmentalization in Yeast by Messenger RNA Transport and a Septin Diffusion Barrier

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Science  13 Oct 2000:
Vol. 290, Issue 5490, pp. 341-344
DOI: 10.1126/science.290.5490.341

Abstract

Asymmetric localization of proteins plays a key role in many cellular processes, including cell polarity and cell fate determination. Using DNA microarray analysis, we identified a plasma membrane protein-encoding mRNA (IST2) that is transported to the bud tip by an actomyosin-based process. mRNA localization created a higher concentration of IST2 protein in the bud compared with that of the mother cell, and this asymmetry was maintained by a septin-mediated membrane diffusion barrier at the mother-bud neck. These results indicate that yeast creates distinct plasma membrane compartments, as has been described in neurons and epithelial cells.

An important means of achieving asymmetric protein distributions is through the cytoskeleton-dependent localization of cytoplasmic mRNAs (1). InSaccharomyces cerevisiae, the transcription factor Ash1p accumulates in the daughter cell nucleus, where it represses mating-type switching (2, 3). The asymmetric distribution of Ash1p is created through the transport ofASH1 mRNA to the bud tip by an actomyosin-driven mechanism (4, 5). Localization of ASH1 mRNA requires at least three proteins that are physically associated withASH1 mRNA: Myo4p (She1p), the myosin motor that transportsASH1 mRNA along actin filaments to the bud tip; She3p, an adapter that mediates the association between Myo4p and ASH1mRNA; and She2p, which is required for the She3p-Myo4p complex to bindASH1 mRNA (6–8). Both She3p and Myo4p localize to the bud tip before ASH1 mRNA expression (9), raising the possibility that other mRNAs are transported in yeast.

To discover other potential localized mRNAs, we identified transcripts that associate with She2p, She3p, and Myo4p using a whole-genome analysis (Fig. 1A). Each of these three proteins was immunoprecipitated from cell extracts using a Myc-epitope tag. Associated RNA was eluted and amplified by reverse transcription followed by polymerase chain reaction (RT-PCR), and the products were fluorescently labeled. An immunoprecipitate from an untagged strain served as a comparative control, and the RT-PCR product from this immunoprecipitate was labeled with a second fluorescent dye. The relative amounts of yeast mRNAs in the She protein versus control immunoprecipitations were determined by hybridization to a DNA microarray containing all S. cerevisiae open reading frames.ASH1 mRNA was enriched in all three immunoprecipitates (2.9-, 2.0-, and 2.2-fold compared with control immunoprecipitates for She2p, She3p, and Myo4p, respectively), validating this approach for identifying other localized mRNAs. Other transcripts that showed a similar enrichment to ASH1 mRNA (10) were then analyzed by fluorescence in situ hybridization (5). One of these transcripts, IST2 (increased sodium tolerance) (11), showed a localization pattern at the bud tip (Fig. 2A). However, in contrast toASH1 mRNA, which is expressed only in late anaphase,IST2 mRNA was localized to the bud tip throughout the cell cycle (Fig. 2A).

Figure 1

The IST2 mRNA is associated with She2p, She3p, and Myo4p. (A) Schematic of the DNA microarray procedure used to identify RNAs associated with She2p, She3p, and Myo4p. Tagged strains are SHE2-13xMYC,SHE3-13xMYC, or MYO4-13xMYC in W303 background and were generated as described (8). Proteins were immunoprecipitated (8), RNA fractions were amplified by RT-PCR, labeled, and analyzed by a DNA microarray (24). (B) Northern analysis (8) of IST2 mRNA confirms that IST2 mRNA is enriched in immunoprecipitates of She2p, She3p, and Myo4p.

Figure 2

Localization of IST2 mRNA to the distal tip of buds requires the She proteins. (A) IST2mRNA was analyzed in wild-type W303 cells by fluorescence in situ hybridization (5). IST2 mRNA was detected at the distal tip of buds and at the presumptive bud site in unbudded cells. Differential interference contrast (DIC) microscopy reveals the locations of cells and buds. (B) IST2 mRNA was analyzed in wild-type and sheΔ strains (W303 background) by fluorescence in situ hybridization (5).IST2 mRNA is distributed throughout the mother cell and bud in she2Δ and myo4Δ cells; she3Δ and she4Δ cells display a similar IST2 mRNA distribution as she2Δ and myo4Δ (16). In bni1Δ cells, IST2 mRNA is localized to the neck region. Quantitative analysis revealed thatIST2 mRNA was asymmetrically localized to the bud in more than 95% of wild-type (W303) cells, whereas in examining over 200 she2Δ, she3Δ, myo4Δ, orshe4Δ cells, an asymmetric distribution of IST2mRNA in the bud was never detected. Most (75%) of bni1Δ cells contained IST2 mRNA at the neck; the remaining 25% of cells showed no localized IST2 mRNA.

To determine whether IST2 mRNA is localized using a mechanism similar to that of ASH1 mRNA, we further tested for a physical association between IST2 mRNA and the She proteins and examined whether localization is disrupted in cells deleted of the SHE genes. Quantitative Northern analysis revealed that IST2 mRNA, like ASH1 mRNA (8), immunoprecipitated with She2p, She3p, and Myo4p (Fig. 1B), confirming the DNA microarray result. Moreover, inshe2Δ, she3Δ, she4Δ, andmyo4Δ cells, IST2 mRNA was distributed throughout the mother and daughter cell (Fig. 2B). Deletion ofBNI1, a gene encoding an actin regulatory protein (12), caused mislocalization of IST2 mRNA to the neck, as was observed for ASH1 mRNA (4,5). Thus, IST2 mRNA localization requires the same protein components as ASH1 mRNA, suggesting that actomyosin-based transport of mRNAs to the distal tip functions throughout the cell cycle.

Expression of a green fluorescent protein Ist2p fusion protein (GFP-Ist2p) from the inducible GAL1 promoter resulted in a fluorescent signal at the plasma membrane (Fig. 3), consistent with sequence predictions indicating the presence of several transmembrane domains in Ist2p (11). GFP-Ist2p localized to the mother cell in small-budded cells, but localized to the bud in medium- and large-budded cells (Fig. 3A). The localization of GFP-Ist2p to the bud required the same proteins needed to localize IST2 mRNA. In cells deleted of SHE2, SHE3, SHE4, orMYO4, GFP-Ist2 was localized to the mother cell even in large-budded cells, whereas bni1Δ cells contained GFP-Ist2p in both mother cell and bud (Fig. 3B). Thus, the asymmetric distribution of Ist2p to the bud is mediated by the localization ofIST2 mRNA to the distal tip of the bud.

Figure 3

Asymmetric distribution of GFP-Ist2p in the plasma membrane of buds requires the She proteins. (A) GPF-IST2 was expressed from the galactose promoter in wild-type cells (25). GFP-Ist2p was observed in the plasma membrane of the mother cell in unbudded and small-budded cells but was localized to the bud in medium- and large-budded cells (indicated by stars). DIC microscopy reveals the locations of cells and buds. (B) Expression of GFP-Ist2p in wild-type andsheΔ strains reveals that the asymmetric distribution of Ist2p in large-budded cells requires the She proteins. she3Δ andshe4Δ cells show a distribution of GFP-Ist2p similar to that of she2Δ and myo4Δ (16). Quantification of the fluorescence intensities in the mother cell and bud revealed that GFP-Ist2p was enriched 3.5-fold in the bud in W303; 2.2-fold in the mother cell in she2Δ; 2.6-fold in the mother cell in she3Δ; 2.1-fold in the mother cell inmyo4Δ; 1.6-fold in the mother cell in she4Δ; and 1.1-fold in the bud in bni1Δ (n = 10).

Although GFP-Ist2p was distributed uniformly throughout the bud, the fluorescence signal abruptly diminished at the neck between the mother and daughter cells (Figs. 3 and 4B). Two possible mechanisms could account for the lack of Ist2p signal beyond the neck: Ist2p is anchored in the plasma membrane of the bud, or Ist2p is mobile in the membrane but is prevented from diffusing into the mother cell by a barrier at the neck. To distinguish between these two possibilities, we determined the mobility of GFP-Ist2p within the plasma membrane by fluorescent recovery after photobleaching (FRAP). To eliminate recovery due to newly synthesized protein, GFP-Ist2p was expressed from the inducible GAL1 promoter. Expression was turned off by adding dextrose. A segment of the plasma membrane of a large bud was photobleached for 1 s, and recovery was monitored every 2 s. The fluorescent signal recovered within 9 s (Fig. 4A), indicating that GFP-Ist2p can diffuse within the plasma membrane of the daughter cell and suggesting that a diffusion barrier at the neck restricts Ist2p to the bud (13).

Figure 4

The GFP-Ist2p fusion protein can diffuse within the plasma membrane of the bud and requires a septin-based diffusion barrier at the neck to maintain asymmetric localization. (A) GFP-Ist2p was expressed in wild-type cells, and the expression was then turned off with dextrose. Large-budded cells containing an asymmetric distribution of GFP-Ist2p were subjected to a 1-s photobleach of the GFP signal in the bud (26). Time-lapse images were taken before the bleach (0 s), and immediately after photobleaching, to assess the recovery of the fluorescence signal in the photobleached area. Twenty large buds were examined, and all showed a recovery of fluorescence signal in the photobleached area similar to that in surrounding areas within 15 s (26). (B) Maintenance of bud-localized GFP-Ist2p requires the septins. GFP-Ist2p was expressed from the galactose promoter in wild-type (A364) andcdc12-6 cells (a temperature-sensitive mutation in one of the septin genes) at the permissive temperature (24°C). Protein expression was turned off by dextrose, and the cultures were then shifted to the restrictive temperature (37°C). The shift in temperature had little effect of GFP-Ist2p in wild-type cells but caused a dramatic decrease in the asymmetric localization of GFP-Ist2p in the septin mutant cell line (27).

One potential candidate for a diffusion barrier is the septin-based ring filament that forms at the neck during bud growth. The septins are required for cytokinesis, although their precise biological roles are not well defined (14). The localization ofIST2 mRNA and protein was examined in cells containing a temperature-sensitive mutation (cdc12-6) in the septin gene, CDC12 (15). At both permissive (24°C) and restrictive (37°C) temperatures, IST2 mRNA was localized to the distal tip of the bud in cdc12-6 cells (16). To determine whether septins affect GFP-Ist2p localization, GFP-Ist2p was expressed at the permissive temperature, protein production was turned off by the addition of dextrose, and then the culture was shifted to the restrictive temperature for 10 min to disassemble the septins (Fig. 4B). At the permissive temperature, 90% of the large-budded wild-type cells displayed an asymmetric distribution of GFP-Ist2p, whereas 52% of the large-buddedcdc12-6 cells contained GFP-Ist2p, predominantly in the bud. Incubation at the restrictive temperature had little effect on GFP- Ist2p localization in wild-type cells (84% of the large-budded cells showed localization to the bud). However, after the 10-min shift to the restrictive temperature, only 12% of the large-buddedcdc12-6 cells contained asymmetrically localized GFP-Ist2p (17). The inability of cdc12-6 cells to maintain the asymmetric distribution of Ist2p at the restrictive temperature suggests that the septins are required to form a barrier at the neck, which prevents Ist2p from diffusing into the mother cell. On the other hand, treatment with the actin-depolymerizing agent, latrunculin-A, did not affect the bud-localized GFP-Ist2p, indicating that actin is not required to maintain GFP-Ist2p in the bud (17). The reduction of asymmetrically localized GFP-Ist2p in cdc12-6 cells at the permissive temperature may result from an imperfect formation of this barrier even at lower temperatures.

These results suggest that the asymmetric localization of Ist2p is achieved through transport of IST2 mRNA to the bud tip by an actomyosin-driven process similar to that described for ASH1mRNA. Once IST2 mRNA is transported and docked at the bud tip, local translation and secretion presumably deliver Ist2p to the plasma membrane of the bud. During this time, turnover diminishes the levels of Ist2p in the mother cell. Maintenance of Ist2p in the bud also requires a septin-mediated diffusion barrier at the neck. It is still unclear whether the septin neck filaments form this barrier or whether they are necessary for localizing other proteins to the neck that act as the diffusion gates.

These findings reveal that yeast has a specialized cytoskeletal architecture at the neck that creates separate plasma membrane compartments in the mother cell and bud that may be important for polarized growth. Septins also are required to maintain the asymmetric distribution of several soluble proteins, such as Myo2p, Sec3p, and Spa2p, that function in polarized growth in the bud (18). If these proteins are associated with transmembrane proteins, the septin-mediated barricade of membrane diffusion may provide a mechanism for trapping these proteins within the bud. Diffusion barriers also are vital to the function of higher eukaryotic cells. In epithelia, the tight junctions act as a protein and lipid diffusion barrier that maintains cell polarity (19, 20). An actin-based diffusion barrier was also described in the initial segment of axons in neurons (21, 22), and several distinct compartments also have been described in mammalian sperm (23). Very little is known, however, about the components and detailed mechanism of these diffusion barriers in differentiated cells. Genetic approaches in S. cerevisiae make this an excellent model system for understanding how the cytoskeleton can restrict the motion of plasma membrane components and create specialized compartments.

  • * To whom correspondence should be addressed. E-mail: vale{at}phy.ucsf.edu

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