Isolation, characterization, and expression of the gene encoding the beta subunit of the mitochondrial processing peptidase from Blastocladiella emersonii.

A 2.3-kb BamHI-KpnI fragment was isolated from a partial genomic library and shown by nucleotide sequence analysis to contain the entire coding region of the gene encoding the beta subunit of the Blastocladiella mitochondrial processing peptidase (beta-MPP). The predicted beta-MPP protein has 465 amino acids and a calculated molecular mass of 50.8 kDa. S1 nuclease protection assays revealed an intron, 209 bp in size, interrupting the coding region between the putative signal sequence and the mature protein. Northern blot analysis showed that beta-MPP mRNA levels decrease significantly during B. emersonii sporulation, reaching basal levels in the zoospore stage. The amount of beta-MPP protein, determined in Western blots, unlike its mRNA, does not vary significantly throughout the fungal life cycle.

Isolation and characterisation of cAMP-dependent protein kinase from Candida albicans. Purification of the regulatory and catalytic subunits.

cAMP-dependent protein kinase (PKA) from Candida albicans yeast cells was isolated and characterised. Structural parameters of the holoenzyme and those of its subunits suggested that C. albicans PKA is a tetramer of 287 kDa composed of two regulatory (R) subunits of 64 kDa and two catalytic (C) subunits of unusually large molecular mass of 78 kDa. The apparent Km for ATP and Kemptide were 30 microM and 60 microM respectively. The [A]0.5 for cAMP activation was 150 nM with a Hill coefficient of 1.6. The holoenzyme undergoes autophosphorylation on the R subunit, a characteristic of the type-II R subunits. Photoaffinity labeling with 8-azido-[32P]cAMP of crude extracts from yeast and mycelial cells strongly suggests that only one type of R subunit is present in the fungus. The R subunit was purified to apparent homogeneity as a protein of 64 kDa. A highly specific polyclonal antiserum raised against the purified protein immunoprecipitated a 64-kDa protein from crude extracts, indicating that the purified R subunit very probably represents the native form of the protein. The 78-kDa form of the C subunit was detected in crude extracts and in Mono Q Sepharose column fractions with heterologous anti-C Ig. It could be isolated by cAMP treatment of the holoenzyme immunoprecipitated from crude extracts with anti-R serum, but this form could not be purified further. Instead, a 60-kDa protein with the main characteristics of C subunit was purified to near homogeneity from soluble extracts of yeast cells. Evidence is presented that this protein very probably derives from the 78-kDa form by proteolytic degradation.

Expression of the groESL operon is cell-cycle controlled in Caulobacter crescentus.

The Caulobacter crescentus groESL operon was cloned, sequenced and found to be homologous to previously described groES and groEL genes and proteins. The size of the groESL-specific transcript (2.3 kb) suggested that groES and groEL of C. crescentus are organized in a bicistronic operon. Heat-shock induction of groESL mRNA is not transient, high levels of the transcript can be observed after 2 h at 40 degrees C. Primer extension experiments showed that transcription initiated at two sites. Only the start site closer to the groES coding region was highly induced during heat shock. The promoter corresponding to the heat-shock-inducible transcript has -10 and -35 regions very similar to Escherichia coli sigma 32 promoters. At normal temperatures, transcription of the groESL operon is cell-cycle controlled and both transcripts increase co-ordinately in pre-divisional cells. Transcription fusions with a lacZ reporter gene and deletions within the promoter region of the groESL operon have shown that no sequences upstream of the heat-shock promoter are necessary for temporal control. An 11 bp inverted repeat, located between the heat-shock promoter and the translation start site of groES and very similar to inverted repeats found in front of several heat-shock genes of other bacteria, may play a role in cell-cycle control of C. crescentus groESL expression.