Regulation of the gene encoding translation elongation factor 3 during growth and morphogenesis in Candida albicans.

The level of the TEF3 mRNA, which encodes the fungal-specific translation elongation factor 3 (EF-3), was measured during the yeast-to-hyphal transition in Candida albicans. In contrast to a previous report, TEF3 mRNA levels were shown to change during dilution into fresh medium, increasing only transiently when dimorphism was induced by either (i) an increase in growth temperature (from 25 degrees C to 37 degrees C) combined with the addition of 10% (v/v) bovine calf serum to the medium, or (ii) an increase in growth temperature (from 25 degrees C to 37 degrees C) combined with an increase in the pH of the medium (from pH 4.5 to 6.5). TEF3 mRNA levels also increased in control cultures under conditions where germ tubes were not formed, but they remained elevated in contrast to cultures undergoing morphological changes. TEF3 mRNA levels were not significantly affected by heat-shock, but were tightly regulated during batch growth of the yeast form, reaching maximal levels in exponential phase. Therefore, the changes in TEF3 expression that accompany the dimorphic transition in C. albicans appear to reflect the underlying physiological changes that occur during morphogenesis and are not a response to morphogenesis per se. For this reason TEF3 mRNA measurement cannot be used as a loading control in Northern analyses of dimorphic gene regulation. Comparison of TEF3 mRNA levels with the abundance of the EF-3 polypeptide indicated that the synthesis of this essential translation factor might be subject to post-transcriptional regulation.

Elongation factor 3 (EF-3) from Candida albicans shows both structural and functional similarity to EF-3 from Saccharomyces cerevisiae.

As with many other fungi, including the budding yeast Saccharomyces cerevisiae, the dimorphic fungus Candida albicans encodes the novel translation factor, elongation factor 3 (EF-3). Using a rapid affinity chromatography protocol, EF-3 was purified to homogeneity from C. albicans and shown to have an apparent molecular mass of 128 kDa. A polyclonal antibody raised against C. albicans EF-3 also showed cross-reactivity with EF-3 from S. cerevisiae. Similarly, the S. cerevisiae TEF3 gene (encoding EF-3) showed cross-hybridization with genomic DNA from C. albicans in Southern hybridization analysis, demonstrating the existence of a single gene closely related to TEF3 in the C. albicans genome. This gene was cloned by using a 0.7 kb polymerase chain reaction-amplified DNA fragment to screen to C. albicans gene library. DNA sequence analysis of 200 bp of the cloned fragment demonstrated an open reading frame showing 51% predicted amino acid identity between the putative C. albicans EF-3 gene and its S. cerevisiae counterpart over the encoded 65-amino-acid stretch. That the cloned C. albicans sequence did indeed encode EF-3 was confirmed by demonstrating its ability to rescue an otherwise non-viable S. cerevisiae tef3:HIS3 null mutant. Thus EF-3 from C. albicans shows both structural and functional similarity to EF-3 from S. cerevisiae.

Candida albicans and three other Candida species contain an elongation factor structurally and functionally analogous to elongation factor 3.

A cell-free poly(U)-dependent translation elongation system from Candida albicans is ATP-dependent due to the presence of an elongation factor 3 (EF3)-like activity. Saccharomyces cerevisiae ribosomes added to a C. albicans postribosomal supernatant (PRS) supported poly(U)-dependent elongation, suggesting that the C. albicans lysate contained a soluble translation factor functionally analogous to the S. cerevisiae translation factor EF-3. The presence of EF-3 in C. albicans was confirmed by Western blotting using an antibody raised against S. cerevisiae EF-3. This antibody was also used to screen a selection of Candida species, all of which possessed EF-3 with molecular mass in the range of 110-130 kDa.

Efficient translation of synthetic and natural mRNAs in an mRNA-dependent cell-free system from the dimorphic fungus Candida albicans.

An mRNA-dependent cell-free translation system has been developed from the human pathogenic fungus Candida albicans using either S30 or S100 lysates prepared from glass-bead-disrupted whole cells. Translation of the synthetic template poly(U) in this system is highly efficient at temperatures up to 37 degrees C and is ATP-dependent. Studies using a range of elongation-specific inhibitors suggest that the mechanism of translational elongation in C. albicans is similar to that of another yeast, Saccharomyces cerevisiae. A micrococcal-nuclease-treated C. albicans S100 lysate was able to translate exogenously-supplied homologous mRNAs, and a range of heterologous natural mRNAs, using an initiation mechanism that is inhibited by the antibiotic edeine and the 5' cap analogue 7-methylguanosine 5'-monophosphate (m7GMP). As with cell-free lysates prepared from S. cerevisiae, the C. albicans lysate is unable to initiate translation upon natural mRNAs at temperatures above 20 degrees C.

The substrate specificity of protein kinases which phosphorylate the alpha subunit of eukaryotic initiation factor 2.

The alpha subunit of eukaryotic protein synthesis initiation factor (eIF-2 alpha) is phosphorylated at a single serine residue (Ser51) by two distinct and well-characterized protein kinase, the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI). The sequence adjacent to Ser51 is rich in basic residues (Ser51-Arg-Arg-Arg-Ile-Arg) suggesting that they may be important in the substrate specificity of the two kinases, as is the case for several other protein kinases. A number of proteins and synthetic peptides containing clusters of basic residues were tested as substrates for HCR and dsI. Both kinases were able to phosphorylate histones and protamines ar multiple sites as judged by two-dimensional mapping of the tryptic phosphopeptides. These data also showed that the specificities of the two kinases were different from one another and from the specificities of two other protein kinases which recognise basic residues, cAMP-dependent protein kinase and protein kinase C. In histones, HCR phosphorylated only serine residues while dsI phosphorylated serine and threonine. Based on phosphoamino acid analyses and gel filtration of tryptic fragments, dsI was capable of phosphorylating both 'sites' in clupeine Y1 and salmine A1, whereas HCR acted only on the N-terminal cluster of serines in these protamines. The specificities of HCR and dsI were further studied using synthetic peptides with differing configurations of basic residues. Both kinases phosphorylated peptides containing C-terminal clusters of arginines on the 'target' serine residue, provided that they were present at positions +3 and/or +4 relative to Ser51. However, peptides containing only N-terminal basic residues were poor and very poor substrates for dsI and HCR, respectively. These findings are consistent with the disposition of basic residues near the phosphorylation site in eIF-2 alpha and show that the specificities of HCR and dsI differ from other protein kinases whose specificities have been studied.

Efficient translation of the UAG termination codon in Candida species.

Clinical isolates of the dimorphic fungus Candida albicans encode a tRNA that, in a cell-free translation system prepared from the yeast Saccharomyces cerevisiae, efficiently translates the amber (UAG) termination codon. Unusually, the efficiency of this UAG read-through in the heterologous cell-free system is not further enhanced by polyamines. The suppressor tRNA is also able to efficiently translate the UAG codon in the rabbit reticulocyte cell-free system and with efficiencies approaching 100% in a homologous (C. albicans) cell-free system. That the suppressor tRNA is nuclear-encoded is demonstrated by the lack of activity in purified C. albicans mitochondrial tRNAs. Finally, UAG suppressor tRNA activity is also demonstrated in three other pathogenic Candida species, C. parapsilosis, C. guillermondii and C. tropicalis. These results suggest that some, but not all, Candida species have evolved an unusual nuclear genetic code in which UAG is used as a sense codon.

Structure and phosphorylation of eukaryotic initiation factor 2. Casein kinase 2 and protein kinase C phosphorylate distinct but adjacent sites in the beta-subunit.

Eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes can be phosphorylated on its beta-subunit by two different protein kinases, protein kinase C and casein kinase 2. Phosphorylation by these kinases is additive, suggesting that they phosphorylate different sites (serine residues) in eIF-2 beta. Two-dimensional peptide mapping of the phosphopeptides generated from labelled eIF-2 beta by digestion with trypsin, cyanogen bromide or Staphylococcus aureus V8 proteinase showed that protein kinase C and casein kinase 2 phosphorylated distinct and different sites in this protein. This conclusion was supported by the results of analysis of the phosphopeptides on reverse-phase chromatography. Analysis of the phosphopeptides derived from eIF-2 beta labelled by both kinases together strongly suggested that the sites labelled by protein kinase C and casein kinase 2 are adjacent in the primary sequence. These data are discussed in the light of the present understanding of the sequence specificity of the kinases. Rat liver eIF-2 beta was also found to be a substrate for protein kinase C and casein kinase 2, which were again shown to label different serine residues.

Structure and regulation of eukaryotic initiation factor eIF-2. Sequence of the site in the alpha subunit phosphorylated by the haem-controlled repressor and by the double-stranded RNA-activated inhibitor.

Eukaryotic protein synthesis initiation factor 2 (eIF-2) can be phosphorylated on its alpha subunit by two well-characterised protein kinases, termed the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI). Phosphorylation of eIF-2 by these kinases is thought to be important in the regulation of peptide-chain initiation. We report the location of the serine residue in the alpha subunit, which is phosphorylated by both these enzymes. Limited tryptic digestion and subsequent cyanogen bromide treatment of rat liver eIF-2 phosphorylated by HCR yielded one major phosphopeptide. This peptide had the sequence Ile-Leu-Leu-Ser-Glu-Leu-Ser(P)-Arg-Arg. The same major phosphopeptide was obtained from rabbit reticulocyte eIF-2 phosphorylated by HCR or dsI as judged by its behaviour on two-dimensional mapping and reverse-phase chromatography. In all cases the phosphorylated residue was found to be serine-7, and not serine-4, of the above sequence as determined from sequence analysis and by subdigestion of the peptide with Staphylococcus aureus V8 proteinase.

Eukaryotic initiation factor 2 from rat liver: no apparent function for the beta-subunit in the formation of initiation complexes.

Eukaryotic protein synthesis initiation factor 2 (eIF-2) from rat liver has been resolved into two subfractions by anion-exchange chromatography on DEAE-cellulose. One of these contained all three components (eIF-2 alpha, eIF-2 beta, eIF-2 gamma) characteristic of mammalian eIF-2, whilst the other fraction contained only two. By a number of criteria these were shown to be eIF-2 alpha and eIF-2 gamma. The absence of eIF-2 beta from this fraction was not due to its proteolytic degradation during purification since it was unaffected by the inclusion of a range of proteinase inhibitors in the isolation media. The properties of eIF-2 containing or lacking eIF-2 beta have been directly compared. It was found that eIF-2 beta was not required for the binding of guanine nucleotides to eIF-2 or for formation of ternary initiation complexes with GTP and the initiator tRNA. eIF-2 lacking eIF-2 beta was able to form 40 S initiation complexes and the presence of eIF-2 beta was also unnecessary for the stimulation of eIF-2 activity by the recycling factor, eIF-2B. Some of these findings are at variance with previous reports in which eIF-2 beta was removed proteolytically. The role of eIF-2 beta in the overall physiological function of eIF-2 remains to be elucidated.