Ralph Pries

Amino acid-dependent Gcn4p stability regulation occurs exclusively in the yeast nucleus.

ABSTRACT The c-Jun-like transcriptional activator Gcn4p controls biosynthesis of translational precursors in the yeast Saccharomyces cerevisiae. Protein stability is dependent on amino acid limitation and cis signals within Gcn4p which are recognized by cyclin-dependent protein kinases, including Pho85p. The Gcn4p population within unstarved yeast consists of a small relatively stable cytoplasmic fraction and a larger less stable nuclear fraction. Gcn4p contains two nuclear localization signals (NLS) which function independently of the presence or absence of amino acids. Expression of NLS-truncated Gcn4p results in an increased cytoplasmic fraction and an overall stabilization of the protein. The same effect is achieved for the entire Gcn4p in a yrb1 yeast mutant strain impaired in the nuclear import machinery. In the presence of amino acids, controlled destabilization of Gcn4p is triggered by the phosphorylation activity of Pho85p. A pho85Δ mutation stabilizes Gcn4p without affecting nuclear import. Pho85p is localized within the nucleus in the presence or absence of amino acids. Therefore, there is a strict spatial separation of protein synthesis and degradation of Gcn4p in yeast. Control of protein stabilization which antagonizes Gcn4p function is restricted to the nucleus.

HARO7 Encodes Chorismate Mutase of the Methylotrophic Yeast Hansenula polymorpha and Is Derepressed upon Methanol Utilization

The HARO7 gene of the methylotrophic, thermotolerant yeast Hansenula polymorpha was cloned by functional complementation. HARO7 encodes a monofunctional 280-amino-acid protein with chorismate mutase (EC 5.4. 99.5) activity that catalyzes the conversion of chorismate to prephenate, a key step in the biosynthesis of aromatic amino acids. The HARO7 gene product shows strong similarities to primary sequences of known eukaryotic chorismate mutase enzymes. After homologous overexpression and purification of the 32-kDa protein, its kinetic parameters (k(cat) = 319.1 s(-1), n(H) = 1.56, [S](0.5) = 16.7 mM) as well as its allosteric regulatory properties were determined. Tryptophan acts as heterotropic positive effector; tyrosine is a negative-acting, heterotropic feedback inhibitor of enzyme activity. The influence of temperature on catalytic turnover and the thermal stability of the enzyme were determined and compared to features of the chorismate mutase enzyme of Saccharomyces cerevisiae. Using the Cre-loxP recombination system, we constructed mutant strains carrying a disrupted HARO7 gene that showed tyrosine auxotrophy and severe growth defects. The amount of the 0.9-kb HARO7 mRNA is independent of amino acid starvation conditions but increases twofold in the presence of methanol as the sole carbon source, implying a catabolite repression system acting on HARO7 expression.

Polyadenylation of rRNA- and tRNA-based yeast transcripts cleaved by internal ribozyme activity

Abstract Polyadenylation, an important step in 3′ end-processing of mRNA in eukaryotes, results in a poly(A) tail that ensures RNA transport into the cytoplasm and subsequent translation. Addition of a poly(A) tail is restricted to transcripts that are synthesized by RNA polymerase II. Here, we demonstrate that the 3′ ends of yeast transcripts based on rRNA and tRNA, respectively, can be polyadenylated in vivo. The transcripts were modified by insertion of a self-cleaving hammerhead ribozyme sequence in the corresponding gene. Both the rDNA-based transcript and the tRNA transcript were cleaved efficiently by the hammerhead ribozyme, resulting in two stable cleavage products. The 5′ cleavage product was found to be polyadenylated in both cases. This demonstrates that, in yeast, transcripts that are usually synthesized by RNA polymerase I or III can be polyadenylated if the 3′ end of the transcript has been generated independently by a ribozyme.

Molecular Biology of Fungal Amino Acid Biosynthesis Regulation

Amino acids are essential precursors for the ribosomal biosynthesis of proteins. In addition, amino acids are used as precursors of nonribosomal synthetic products including such important pharmaceutically relevant secondary metabolites as the β-lactam antibiotics or their derivatives of fungi (Brakhage 1998; see also Chap. 16, this Vol.). Most fungal cells prefer to acquire the 20 different amino acids for translation by uptake from the diet. Amino acid uptake primarily depends on the nutritional conditions and requires appropriate sensors and uptake systems. When the required amino acid is present in the cultivation medium, no further specific enzyme activities are needed. Numerous fungi are also able to produce and secrete proteases to explore additional nutritional sources. Induction of those activities might require starvation conditions (e.g., for nitrogen) as well as the presence of extracellular protein. Secreted proteases permit the extracellular degradation of proteins and, therefore, the production of extracellular amino acids (Ogrydziak 1993; Pavlukova et al. 1998). This is especially required in an environment lacking further nitrogen or carbon sources.

Nuclear import of yeast Gcn4p requires karyopherins Srp1p and Kap95p

The yeast transcription factor Gcn4p contains two stretches of amino acid residues, NLS1 and NLS2, which are independently able to relocate the cytoplasmic protein chorismate mutase into the nucleus. Only NLS2 is conserved among fungi. A truncated version of CPCA (the counterpart of Gcn4p in Aspergillus nidulans), which lacks the conserved NLS, accumulates in the cytoplasm instead of the nucleus. Nuclear uptake mediated by the NLS1 of Gcn4p is impaired by defects in genes for several different karyopherins, whereas NLS2-dependent nuclear import specifically requires the α-importin Srp1p and the β-importin Kap95p. Yeast strains that are defective in either of these two karyopherins are unable to respond to amino acid starvation. We have thus identified Gcn4p as a substrate for the Srp1p/Kap95p transport complex. Our data suggest that NLS2 is the essential and specific nuclear transport signal; NLS1 may play only an unspecific or accessory role.