P ROYECTOS EN CURSO

Transcriptional activation by H AP2/3 /4/5 is observed when cells are shifted from a fermentable carbon source to a non-fermentable one, ie. in the absence of CCR. The activity of the complex has been shown to _{be induced by carbon source, as} S. cem;isiae s trains containing mutated hap2, hap3 or hap4 genes are incapable of growth on non­ fermentable carbon sources such as lactate (Forsburg & G uarente, 1 989) .

The exact mechanism by which the carbon source signal i s transduced to the cells i s not yet known. The expression of the hap2 and hap3 genes is constitutive, while transcription o f hap4 i s induced upon a switch t o a non-fermentable carbon source (Forsburg & Guarente,

1 989) . Therefore, the activity of the complex is regulated by HAP4, although this is not always the case. For example, transcription of the 5. tereiJZsiae ./1SN 1 gene is activated by HAP2/3/5, even in a hap4 mutant strain (Brakhage et al. , 1 999). The HAP2/3/5 complex was therefore suggested to activate gene expression by remodelling the chromatin structure, while HAP4 can act as a direct transcriptional activator.

The activity of the complex is also influenced by heme, as is shown by the 1 0-fold decrease in HAP2/3/4/5 activity at UAS2 in heme-deficient cells, compared to wildtype cells. Thus respiratory genes activated by HAP2/3/4/5 are also considered to be sensitive to the availability of oxygen, as it is required in heme biosynthesis (Figure 1 .6) (Forsburg &

Guarente, 1 989) .

1.9.3 THE SUBUNITS OF THE HAP2/3/4/5 COMPLEX

The HAP2/3/5 subunits are necessary for DNA-binding, and accomplish this as a heteromeric complex. All of the subunits therefore contain specific domains for DNA­ binding and subunit association. Dimerisation of HAP3 and HAPS occurs initially, faciliated by the conserved histone motifs contained within the proteins. H AP2 then associates with HAP3/5. The sequential formation of the heteromeric complex occurs without DNA binding, although this event stabilises the protein-protein interactions within the complex (Brakhage et al., 1 999) . Lastly, the H A P4 protein interacts with the complex.

Chapter 1 This protein contains an essential, highly acidic domain at its carboxyl terminus which is functionally homologous to the acidic transcriptional activation domain of the GAL4 protein (Olesen & Guarente, 1 990) .

1.9.4 THE SUBUNITS OF THE ANCF

H omologues of the HAP2/3/5 subunits have been found in /1. nidulans; H APB, H APC and HAPE, respectively. These subunits have been overexpressed in E. coli and found to form a heteromeric complex in IJi!ro (Brakhage el al. , 1 999) .

an A. nid11lam homologue to the S. cenn;isiae HJ.\ P4 protein has not been found as yet, analysis of fungal H A PS homologues (eg. H.A PE) have revealed the presence of a domain which is necessary for interaction with the Hr\ P4 protein. Also, electrophoretic mobility s hift assays (E ISA's) with /1. nidulans nuclear extracts and CCAA T-containing probes generate two complexes. One band is analogous to the heteromeric complex formed by H APB/C/E (approximately 87 kDa), the other is approximately 1 20 - 1 30 kDa (eg. Hr\PB/C/E plus another protein) (Brakhage el al., 1 999) . Therefore, there is evidence for an A. nidulans homologue of H AP4. Interestingly, autoregulation of the A CF complex was recently reported to occur via its HAPB subunit (Steidl el al., 200 1 ) .

1.9.4 ASSEMBLY OF THE HAPB/C/E COMPLEX (ANCF)

A model for the assembly of the HAPB/C/E complex is shown in Figure 1 .9. Si.mi.lar to the H A P2/3/5 complex, HAPC and H A PE interact initially and form a dimer; HAPB tl1en assooates. D to the CCAAT element by the heteromeric complex is then possible.

Unlike the H A P2/3/5 complex, activity of the AnCF does not appear to be an absolute requirement for transcriptional activation in A. nidulam, as strains deficient in hapb, hapc or

hape are capable of growtl1 on non-fermentable carbon sources (Brakhage et al., 1 999) . This result is not surprising, as many of the genes regulated by CCAAT-binding complexes are involved in respiratory processes, and A. nidulans is an obligate aerobe, unlike S. cerevisiae.

HAPC

•

'

•

Figure 1.9 Assembly Of The AnCF. Proteins HAPC and HAPE interact v1a conserved histone motifs to form a climer. HAPB then associates, and the heteromeric AnCF is formed. A putative fourth subunit, homologous to HAP4, also interacts with the complex. The complex interacts specifically with the CCAA T element within the target gene promoter.

Chapter 1

1.10 AIMS OF THE PROJECT

The viability of an A. nidu!am strain deficient in cytochrome c provided the impetus for this project. Other cytochrome c deficient mutants had been generated in the past, but none in an obligate aerobe. Therefore, the elucidation of the alternate energy-yielding processes used by this s train was of great interest.

The observation of putative regulatory elements within the A. nidu!am cytochron1e t· gene

(rytA) also raised the intriguing question as to why the expression of a seemingly essential gene would be regulated.

ny fmdings regarding the regulation of respiratory processes in this fungus would have important implications, given the extensive use of ftlamentous fungi in commerical applications

Determining the activity of an alternative respiratory pathway in A. mdu/am, and possible mechanisms which may regulate this activity, would also be an important finding o f this

study. Alternative respiratory pathways have recently been suggested to play an in1portant role in enabling pathogenic fungi to overcome host defence mechanisms upon invasion. Thus, the .AOX protein has been suggested to be an attractive target for antifungal therapy. Investigation into tl1e activity of tl1e alternative respiratory pathway in A. mdulam would tl1erefore be extremely beneficial, given the predominance of tlus fungus as an opportunistic pathogen.

Thus the aims of the project were:

1 . Characterisation of the cytochrome c-deficient mutant of A. nidulans, to compare the growth of t!Us strain witl1 wild type strains of A. nidulans and other cytochrome c deficient mutants (Chapter 3) . Spectral analysis was required to confirm the absence of detectable cytochrome c in tl1ese strains. 1 he use of the alternative respiratory pathway and fermentative metabolism in the rycA- strains was also investigated (Chapter 3) .

2. Characterisation o f the alternative oxidase gene of A. nidu/ans (Chapter 4), wruch may encode tl1e protein involved in the alternative respiratory pathway. Expression of the A. nidu/ans AOX under varying physiological conditions was also investigated (Chapter 4) .

Chapter 1

3. Functional analysis of d1e A. nidulam cytochrome .- gene promoter, to determine

d1e significance of putative regulatory elements which were identified previously (Chapter 5) . Particular interest was focused on the oxygen regulation of ryt'i\ gene expression, which may be mediated by a HAP1 -like transcription factor.

4. Functional expression of the A. nidulam cytochrome .- gene promoter in S. t'em;isiae

would determine whether yeast proteins are able to regulate rycA gene expression (Chapter 6) . It had been speculated that this was likely, given the similarities in regulatory mechanisms controlling cytochrome .- expresston 111 _{these two}

Chapter 2

CHAPTER 2