The cAMP signal transduction pathway controls many processes in fungi. The Mucor circinelloides pkaR and pkaC genes, encoding the regulatory (PKAR) and catalytic (PKAC) subunit of the cAMP-dependent protein kinase A (PKA), have been cloned recently. Expression analysis during the dimorphic shift and colony morphology suggested a role for PKAR in the control of morphology and branching. Here strain KFA121, which overexpresses the M. circinelloides pkaR gene, was used to quantify growth and branching under different aerobic growth conditions in a flow-through cell by computerized image analysis. An inverse relationship between the pkaR expression level in KFA121 and the hyphal growth unit length was observed in KFA121, suggesting a central role for PKAR in branching. A biochemical analysis of PKAR using antibodies and enzyme assay demonstrated that the level of PKAR is higher in KFA121 under inducing conditions, i.e. in the presence of high glucose, than in the vector control strain KFA89. Measurement of cAMP binding demonstrated a significant increase (two- to threefold) in PKAR level for KFA121 at the time of germ-tube emission in medium containing 10 g glucose l(-1). The level of PKA activity was determined using kemptide in the same crude cell extracts used to determine cAMP binding. Strain KFA121 showed a twofold increase in PKA activity. An excess of free PKAR subunit over PKA holoenzyme was determined using sucrose gradient centrifugation of extracts from KFA89 and KFA121. The data indicate that cAMP-dependent PKA in M. circinelloides might be down-regulated during hyphal-tube emergence and that an increase in PKAR levels results in increased branching.
|Publication status||Published - 2004|