Asc1p interactions and functions known in Saccharomyces cerevisiae

Additionally to encoding for Asc1p, the ASC1 gene belongs to the 5% of yeast genes containing an intron in their open reading frame (ORF). This rather small amount of genes has been described to be responsible for the bulk of mRNA in the cell, which is also reflected in high levels of ASC1 expression, resulting in 330,000 Asc1p molecules per cell

(Ares et al., 1999; Ghaemmaghami et al., 2003). The ASC1 intron consists of 273 nucleotides in the proximity to the 3’ end of the ASC1-ORF and codes for the small

C/D box nucleolar RNA (snoRNA) U24 (Tyc and Steitz, 1989; Maxwell and Fournier, 1995). It is involved in ribosome biogenesis through post-transcriptional site-specific 2’-O-methylation of the 28S rRNA and is the only snoRNA that is required for more than two such modifications (Kiss-László et al., 1998; Schattner et al., 2004).

3.1.2 Asc1p interactions and functions known in Saccharomyces cerevisiae

The exposed positioning of Asc1p on the ribosome and its asymmetric seven-bladed propeller structure is conserved from yeast to human and results in its interaction with a

multitude of proteins and ligands (Gavin et al., 2002; Gavin et al., 2006;

Coyle et al., 2009). For example it has been shown that Asc1p physically interacts with the MAP kinase Slt2p of the Pkc1p cell wall integrity pathway (Breitkreutz et al., 2010). An

Figure 6. The localization of Asc1p on the ribosome (modified from Coyle et al., 2009).

Asc1p resides on the 40S ribosomal subunit near the mRNA exit tunnel in close proximity to helices 39 and 40 of the 18S rRNA. The part of the protein containing the knob-structure faces the ribosome, whereas the remaining part of the protein is accessible.

CHAPTER I 24

additional implication of Asc1p in this pathway is given by the enhanced phosphorylation of Slt2p in the absence of Asc1p (Chasse et al., 2006) and a higher sensitivity of a ∆asc1 strain for the cell wall drugs calcofluor white and zymolyase (Valerius et al., 2007).

Despite the described interaction with PKC in mammalian cells with the Asc1p orthologue RACK1 (Receptor of Activated protein Kinase C), it has been shown for Asc1p in yeast that it specifically influences cell wall integrity near bud sites by a Pkc1p-independent mechanism (Melamed et al., 2010).

Another interaction of Asc1p was described with the mRNA-binding protein Scp160p and furthermore that this interaction is required for the recruitment of Scp160p and its associated messages to the ribosome (Baum et al., 2004). Subsequently an extended Asc1p network (Smy2p, Eap1p, Scp160p and Asc1p; SESA network) has been identified to specifically inhibit the translation initiation of the POM34-mRNA, encoding an integral membrane protein of the nuclear pore complex (Sezen et al., 2009). In agreement, earlier studies have described Asc1p as a translational repressor (Gerbasi et al., 2004). Its first

discovery linked Asc1p to the process of translation initiation in context with heme-deficient growth as its deletion suppressed a hem1^-cyp1^- absence of growth

phenotype (Chantrel et al., 1998). Further evidence for an inhibitory effect of Asc1p on translation initiation is the enhanced phosphorylation of the translation initiation factors eIF4A and eIF2 (Valerius et al., 2007), the latter inhibiting the formation of the ternary complex required for the initiation process (Dever et al., 1992; Voorma et al., 1994).

Additionally, ASC1 genetically interacts with GCN2, encoding the eIF2 kinase. The additional deletion of ASC1 suppresses the absence of growth phenotype of a ∆gcn2 strain on amino acid starved medium (Hoffmann et al., 1999).

Whereas resistance to calcofluor white and the Scp160p interaction with the ribosome have been identified as processes dependent on Asc1p as ribosomal constituent, another described function for Asc1p in cell signaling is suggested to be independent of its ribosome-association (Zeller et al., 2007; Coyle et al., 2009). According to structural investigations via crystallization studies the organization of Asc1p on the ribosomal interface excludes simultaneous binding to the Gα-protein Gpa2p involved in the pathway of invasive and pseudohyphal growth (Coyle et al., 2009). Asc1p has been described as a repressor of this pathway, which serves to alter S. cerevisiae cell morphology in response to changes in nutrient-availabilities (Figure 7) (Mösch et al., 1999; Zeller et al., 2007).

CHAPTER I 25

Limitations in nitrogen and glucose induce the pathway and result in diploid pseudohyphal growth and haploid invasive growth, respectively (Gimeno et al., 1992; Cullen and Sprague, 2000), through the expression of FLO11, coding for a GPI-anchored cell wall flocculin (Lo and Dranginis, 1998; Rupp et al., 1999).

The pathway is divided in two signaling cascades, the cAMP-dependent protein kinase (PKA) pathway and the mitogen-activated protein kinase (MAPK) pathway, which both have been shown to be inhibited by Asc1p (Figure 7) (Zeller et al., 2007).

Figure 7. Scheme of signaling pathway of invasive/pseudohyphal growth. PKA pathway as well as MAPK pathway are illustrated. In the PKA pathway Gpr1p functions as glucose-sensor in the plasma membrane. Upon glucose-limitation it interacts with the heterotrimeric G protein α subunit, Gpa2p, to activate the adenylate cyclase Cyr1p, which in turn elevates cAMP-levels within the cell. cAMP activates the cAMP-dependent protein kinase (PKA), which results in the release of Tpk2p and subsequent phosphorylation of the transcriptional repressor Sfl1p and activator Flo8p to induce FLO11-expression (Pan et al., 2002). The MAPK pathway is induced by glucose-limitation through an unknown sensor, which results in the phosphorylation of Ste20p. The phosphorylation is passed on through the MAPK cascade to the transcription factor Ste12p and its inhibitors Dig1p/Dig2p, resulting in the release of Ste12p and subsequent activation of the transcription factor Tec1p (Elion et al., 1993; Tedford et al., 1997). The activated transcription factors then bind to the FLO11-promoter at Tec1p-binding sites (TCS) or filamentous response-elements (FRE) to induce transcription (Baur et al., 1997; Madhani and Fink, 1997; Madhani and Fink, 1998). The physical interactions of Asc1p and the described phenotypes for a ∆asc1 strain are illustrates in red and green according to Valerius et al., 2007 and Zeller et al., 2007.

CHAPTER I 26

The MAP kinase cascade, including Ste20p, Ste11p, Ste7p, and Kss1p, is activated by glucose through binding to an unknown receptor (Mösch et al., 1996; Mösch et al., 1999).

The inhibitory influence of Asc1p on this pathway was demonstrated through the elevated phosphorylation of the MAP kinase Kss1p when ASC1 is deleted. Additionally, Asc1p-binding to Ste20p was shown (Zeller et al., 2007).

In the second branch of the signaling pathway, glucose binds to the plasma membrane glucose-sensor Gpr1p (Lemaire et al., 2004), which activates the heterotrimeric G protein α subunit Gpa2p. This in turn activates the adenylate cyclase Cyr1p (Colombo et al., 1998;

Kraakman et al., 1999), which leads to increased cellular concentrations of cAMP (Kataoka et al., 1985). Asc1p has been found to interact with the GDP-bound form of Gpa2p as Gβ subunit and inhibits the Gpa2p guanine nucleotide exchange activity, as required for proper inducability of the signaling cascade. Additionally, its physical interaction with Cyr1p was shown and an enhanced Cyr1p-dependent cAMP-production was observed in a ∆asc1 strain background (Zeller et al., 2007).

Contradictory to these findings pointing to an enhanced activity of the signaling pathway of invasive/pseudohyphal growth in a ∆asc1 strain, cells lacking Asc1p are not able to undergo the expected physiological changes in response to outside stimuli. Neither glucose starvation-induced invasive growth nor pseudohyphal differentiation in response to nitrogen-limitation can be observed in ∆asc1 cells (Valerius et al., 2007; Zeller et al., 2007). These growth phenotypes could be traced back to a reduced expression of FLO11, resulting in a drastic reduction in mRNA as well as protein levels of Flo11p in the ∆asc1 strain (Valerius et al., 2007) (Figure 7).