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. 2003 May;23(9):3253-64.
doi: 10.1128/MCB.23.9.3253-3264.2003.

Ste11p, a high-mobility-group box DNA-binding protein, undergoes pheromone- and nutrient-regulated nuclear-cytoplasmic shuttling

Jian Qin  1 , Wenfei Kang, Betty Leung, Maureen McLeod
Affiliations

Ste11p, a high-mobility-group box DNA-binding protein, undergoes pheromone- and nutrient-regulated nuclear-cytoplasmic shuttling

Jian Qin et al. Mol Cell Biol. 2003 May.

Abstract

The high-mobility-group (HMG) box is a conserved DNA-binding domain found in a family of transcription factors that regulate growth and development. One family member, Ste11p, directs sexual differentiation of Schizosaccharomyces pombe by binding specific DNA sequences upstream of genes required for mating and meiosis. Here, we show that Ste11p is a shuttling protein. In growing cells, Ste11p is present in low levels and is pancellular. Mating pheromones and nutrient limitation trigger nuclear accumulation and increased expression of the transcription factor. Several mechanisms likely control Ste11p localization. First, the 14-3-3 protein, Rad24p, binds phosphorylated Ste11p and inhibits its nuclear accumulation. Second, the HMG domain of Ste11p contains a basic cluster nuclear localization signal. Finally, treatment of cells with leptomycin B, an exportin inhibitor, results in the nuclear accumulation of Ste11p. A Ste11p deletion mutation, DeltaC54, mimics the effects of leptomycin B. The C54 region contains no identifiable nuclear export signal but instead is required for biological activity and to stimulate Ste11p target gene expression. These results provide evidence that both nuclear import and export mechanisms operate to regulate cellular localization of an HMG box protein. In addition, they establish a paradigm for the potential role of pheromone/hormone-like polypeptides in cellular localization of this important class of developmental regulators.

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Figures

FIG. 1.
FIG. 1.
Ste11p accumulates in the nucleus of signaling cells prior to and during conjugation. (A) Schematic drawing of cells at visually distinguishable stages of sexual development. Some genes required at several defined stages are indicated on the right. (B) Images of h90 ste11-gfp cells (SPB371) were acquired by confocal microscopy at the indicated times following a shift to nitrogen-free medium. (C) Growing and starved SPB371 cells containing Ste11p-GFP (top panels) were stained with Hoechst 33324 (bottom panels) to visualize the nucleus. (D) Images of the designated nitrogen-starved cells (h+, SPB373; h90 fus1, SPB380; h90 mei3, SPB399) were visualized by fluorescence microscopy.
FIG. 2.
FIG. 2.
Nuclear accumulation of Ste11p-GFP requires nitrogen starvation and pheromone signaling. (A) A montage of h− ste11-gfp cells (SPB394) incubated in the presence (N+) or absence (N−) of a nitrogen source. At the time of the nutritional shift, either synthetic pheromone (P+) or vehicle (P−) was added to the indicated samples. (B) The number of cells displaying nuclear accumulation of Ste11p-GFP was determined as described in Materials and Methods.
FIG. 3.
FIG. 3.
Role of Pat1p in activation of Ste11p. (A) Inactivation of Pat1p kinase bypasses the starvation and pheromone signals for nuclear accumulation of Ste11p. Shown is a montage of h− pat1::ura4 mei2ts cells (right panel; SPB408)) and h− mei2ts cells (left panel; SPB410) expressing Ste11p-GFP. Cells were incubated at 34°C (to inactivate mei2ts) in complete medium. (B) Conjugation and sporulation rates indicate that ste11-AA is an activated allele. The percentage of h90 cells containing either ste11-AA (○; SPB361) or ste11 (•; SPB363) that were able to undergo conjugation and meiosis is indicated. Cells were shifted to nitrogen-free medium. At various times, a portion of the culture was examined to determine the number of single cells, conjugated cells and asci. wt, wild type. (C) Expression of the mei2-lacZ reporter gene in h90 and h+ cells combined with either ste11 or ste11-AA following nitrogen starvation. Cells with the indicated genotypes were shifted to nitrogen-free medium. At various times, a portion of each culture was removed, and β-galactosidase activity was measured with a permeabilized cell assay. Activity is expressed in Miller units (see Materials and Methods). Mean values from three independent samples are shown. Genotypes of the strains used are listed in Table 1. They were SPB368, SPB366, SPB59, and SPB367.
FIG. 4.
FIG. 4.
Ste11p contains specific regions required for nuclear accumulation. (A) Full-length Ste11p (represented by a box) and Ste11p deletion derivatives were fused to GFP. The gray bar represents the HMG domain, with potential bipartite (Bip) and basic cluster (BC) NLS sequences in black. The RKDI and RKDII regions contain Pat1p substrate specificity determinants (22). The fusion proteins were produced from plasmids in h90 (SPB6) and h− (SPB61) cells, and nuclear accumulation was scored in actively growing cells. A positive score indicates that ≥1.0% of the cells accumulated the fusion protein in the nucleus; a negative score indicates that ≤1.0% cells had nucleus-accumulated GFP. (B) A montage illustrating the distribution of Ste11p-GFP (pSte11.102) and C54-GFP (pSte11.60) in growing and starved cells. Cells transformed with plasmids expressing either complete Ste11p (pSte11.102) or a version deleted of 54 carboxyl-terminal residues (pSte11.60) were examined with a microscope. Nuclear accumulation was scored in growing and starved h90 (SPB6) and h− (SPB61) cells. Note that in the starved h90 culture, the subcellular distribution of Ste11p-GFP is regulated so that only a subset of the cells display nuclear accumulation of the protein (see Fig. 1B). In contrast, cells containing the deletion derivative (pSte11.60) accumulate nuclear GFP at all stages of the life cycle. (C) The basic cluster is necessary and sufficient to target Ste11p-GFP to the nucleus. h− cells (SPB61) were transformed with the indicated plasmids. pSte11.118L was fused to GFP via a 75-amino-acid linker sequence (L-GFP).
FIG. 5.
FIG. 5.
LMB treatment causes nuclear accumulation of Ste11p in the absence of nutritional and mating pheromone signals. (A) h90 (SPB371), h+ (SPB373), and h− (SPB375) cells growing in nutrient-rich medium were mock treated or treated with LMB. At the indicated times, a portion of each culture was removed for photography. (B) A graphic representation of the number of cells containing nucleus-accumulated Ste11p-GFP.
FIG. 6.
FIG. 6.
ΔC54 is required for biological activity. (A) A schematic representation of several ste11 alleles expressed from plasmids with the adh promoter. The HMG box is shaded in gray. The basic cluster is illustrated in black. Also indicated are the RKDI and RKDII regions (gray lines). Plasmids containing the deletions fused to GFP were transformed into h90 cells, and the subcellular distribution of the chimeric proteins was determined in growing cells. The cells were scored as indicated in the legend to Fig. 4. Suppression is the ability to reverse the conjugation and sporulation defect of h90 ste11::ura4 cells. a.a., amino acid. (B) Expression of the mei2-lacZ reporter gene in h90 ste11::ura4 cells (SPB162) transformed with the indicated plasmids. pALT2 is the empty vector. pSte11.60 and pSte11.102 are described in Fig. 4. Cells were shifted to nitrogen-free medium. At various times, a portion of each culture was removed, and β-galactosidase activity was measured. Activity is expressed in Miller units (see Materials and Methods). Mean values from three independent samples are shown.
FIG. 7.
FIG. 7.
Expression of Rad24p delays sexual differentiation and causes a defect in nuclear accumulation of Ste11p. (A) Cells transformed with empty plasmid (pALT2) or a plasmid expressing rad24 from the adh promoter (pRad24.1) were shifted to nitrogen-free medium. At various times thereafter, the number of individual cells, conjugating cells, and spore-containing cells was determined. (B) Expression of Rad24p alters accumulation of Ste11p-GFP in the nucleus. h− cells containing empty vector (V) or pRad24.1 (R) were mock treated or treated with LMB for 10 min prior to photography. (C) Quantitation of nucleus-localized Ste11p-GFP from the above experiment. Cells treated with LMB (⧫ and •) or mock treated (▴ and ×) were photographed at the indicated times following the addition of drugs. Cells were counted to determine the percentage containing nuclear fluorescence. (D) Expression of Rad24p inhibits activation of the mei2-lacZ reporter gene in h90 mei2::lacZ cells. SPB162 cells were transformed with empty vector (pALT2) or a plasmid expressing Rad24p (pRAD24.1). Growing transformants were shifted to nitrogen-free medium. At various times, a portion of each culture was removed, and β-galactosidase activity was measured with a permeabilized cell assay. Activity is expressed in Miller units (see Materials and Methods). Mean values from three independent samples are shown.
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