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

Transcription factors belonging to the high-mobility-group (HMG) box superfamily share a conserved DNA-binding domain. Members include Sry, the developmental regulator of sex determination in humans; the related Sox (Sry box) proteins; and the TCF and LEF transcription factors (28, 39). Sequences within the HMG box bend DNA by interacting with the minor groove (43). This property allows HMG box proteins to act as architectural elements specifying the assembly of higher-order nucleoprotein complexes (12, 25). HMG proteins are important for differentiation of diverse cell types, such as those specifying cardiac, neural, and lymphoid tissues (49). Dysfunctional HMG proteins are linked to several disease states, including malignancy (35). Ste11p is a 52-kDa HMG box protein that regulates expression of cell mating type genes required for conjugation and sporulation of Schizosaccharomyces pombe (41). Since Ste11p is structurally related to members of the HMG domain superfamily (28), the molecular mechanisms used to regulate its activity are likely to be of general significance. Yeast cells reproduce vegetatively, but specific conditions can cause cells of opposite mating types to conjugate and form a diploid zygote that is capable of meiosis (5, 8). Conjugation is a complex process initiated by deprivation of nutrients from the growth medium and by pheromone signals. These cause G1 cell cycle delay, cell-cell agglutination and fusion, and karyogamy. The newly formed diploid zygote is capable of meiosis if pheromone signaling and nutrient limitation conditions continue. Pat1p/Ran1p protein kinase (referred to as Pat1p hereafter) has a pivotal function in establishing both competence to differentiate and commitment to the process. During vegetative growth, Pat1p kinase inhibits conjugation and sporulation (26). Nutrient limitation and pheromone signaling cause inactivation of Pat1p (2). Inhibition of the kinase is both necessary and sufficient for differentiation (2, 14, 30), though not all means by which inactivation is accomplished have been described at the molecular level. Genetic and biochemical studies identified Ste11p and Mei2p as substrates for Pat1p (16, 22, 47). Mei2p is an RNA-binding protein (48). Phosphorylation by Pat1p is inhibitory for Mei2p. It converts Mei2p into a substrate for ubiquitin-dependent proteolysis (16) and inhibits its RNA-binding ability (36). In the absence of Mei2p, cells conjugate, and the resulting diploid zygote arrests prior to premeiotic DNA synthesis (3, 37, 48). Thus, Mei2p is not essential for the early steps of sexual differentiation. In contrast, Ste11p is essential for both conjugation and sporulation (38, 41) and is the most immediate downstream target of Pat1p (22). As it does for Mei2p, phosphorylation of Ste11p inhibits its activity. Inactivation of Pat1p is sufficient for robust expression of ste11 (31), since Ste11p is autoregulatory (20, 41). Ste11p binds a specific DNA sequence, the TR box, found upstream of genes required for mating and sporulation. This includes the mating type genes, mei2, and ste11 itself (18, 41). The mating type genes regulate production of cell type-specific pheromones and pheromone receptors. Pheromone communication allows cells of opposite mating types to conjugate and to undergo meiosis (5, 8). Meiosis is caused by activation of mei3 expression. Two mating type proteins, MatPm and MatMc, directly regulate mei3 transcription (44). Mei3p is an inhibitor of Pat1p that is essential for full inactivation of the kinase and commitment to meiosis (26, 27, 45, 50). Thus, Pat1p and Ste11p are elements of a positive feedback loop. The requirement for nutrient limitation in sexual differentiation and its role in expression of ste11 are well established. However, mating pheromone communication is only indirectly implicated in regulation of Ste11p activity. We have proposed that Ste11p activity is directly repressed by Pat1p phosphorylation (22). In this model, inactivation of Pat1p by nutrient limitation leads to accumulation of a nonphosphorylated, activated form of Ste11p. Ste11p autoregulation amplifies the initial starvation signal and causes expression of mating pheromones and receptors. Pheromone signaling further inactivates Pat1p kinase, consequently activating Ste11p and amplifying expression of Ste11p and its downstream targets. Molecular details to support this model are lacking. For instance, it is not known if Ste11p is an in vivo substrate for Pat1p or if Pat1p-specific phosphorylation alters the activity of the transcription factor. Some regulators of Ste11p transcription factor activity are known. For example, cell type-specific expression of M factor, encoded by mfm1, requires an interaction between Ste11p and another HMG box protein, Mat1-Mc. The interaction strengthens Ste11p binding to weak TR boxes (18). Additionally, Rad24p physically interacts with Ste11p and inhibits its activity by an as yet unknown mechanism (16). Rad24p is a 14-3-3 protein (9). Members of the 14-3-3 family coordinate multiple signal transduction pathways. In the fission yeast Schizosaccharomyces pombe, Rad24p functions in the DNA synthesis checkpoint as a cytoplasmic tether for the Cdc25p mitotic activator (9, 23, 24). Rad24p also has a role in conjugation. In its absence, cells conjugate and ste11 is expressed without the need for starvation (9). On the other hand, high-level expression of Rad24p causes a reduction in the steady-state level of both the ste11 transcript and its protein product. Rad24p binds preferentially to Ste11p that has been phosphorylated by Pat1p (16). In support of this observation, the amino acid sequences around both phosphorylation sites resemble the consensus recognition sequence for members of the 14-3-3 protein family. Although Rad24p functions to regulate the localization of some regulatory proteins (23), Rad24p did not appear to alter the localization of Ste11p. Thus, it has been proposed that Rad24p may be important in the transcriptional activation or DNA binding of Ste11p (16). In the present study, evidence is presented that localization of Ste11p is one means of regulating its activity. Ste11p is not constitutively nuclear but accumulates in that compartment during specific stages of the cell's life cycle. Systematic deletion analysis showed that the HMG box of Ste11p contains a functional basic cluster nuclear import signal. Pheromone signaling and nutrient limitation likely control accumulation of Ste11p in the nucleus by inhibiting its crm1 (exportin)-dependent export. An ste11 allele, ste11ΔC54, produces a truncated polypeptide found constitutively localized to the nucleus. The Ste11ΔC54p polypeptide is inactive, indicating that nuclear accumulation of Ste11p is not sufficient for its function. In addition to import and export, the subcellular distribution of Ste11p is regulated by other mechanisms. Contrary to other reports, we found that Rad24p alters the subcellular distribution of Ste11p. Rad24p sequesters Ste11p in the cytoplasm so that it is not available for import into the nucleus.