Molecular characterization of a G protein alpha-subunit-encoding gene from Mucor circinelloides.
Genes encoding the Galpha subunit were cloned from Mucor circinelloides, a zygomycete dimorphic fungus. There are at least four genes that encode for Galpha subunits, gpa1, gpa2, gpa3, and gpa4. The genes gpa1 and gpa3 were isolated and characterized, and their predicted products showed 36%-67% identity with Galpha subunits from diverse fungi. Northern blot analysis of gpa3 showed that it is present in spores and constitutively expressed during mycelium development and during yeast-mycelium and mycelium-yeast transitions. However, during yeast cell growth, decreased levels of mRNA were observed. Sequence analysis of gpa3 cDNA revealed that Gpa3 encodes a polypeptide of 356 amino acids with a calculated molecular mass of 40.8 kDa. The deduced sequence of Gpa3 protein contains all the consensus regions of Galpha subunits of the Galpha(i/o/t) subfamily except the cysteine near the C terminus for potential ADP-ribosylation by pertussis toxin. This cDNA was expressed in Escherichia coli and purified by affinity chromatography. Based on its electrophoretic mobility in SDS-PAGE, the molecular mass of the His6-tagged Gpa3 was 45 kDa. The recombinant protein was recognized by a polyclonal antibody against a fragment of a human Galpha(i/o/t). Furthermore, the recombinant Gpa3 was ADP-ribosylated by activated cholera toxin and [32P]NAD but not by pertussis toxin. These results indicate that in M. circinelloides the Galpha subunit Gpa3 is expressed constitutively during differentiation.
Meza-Carmen
Victor
V
Instituto de Investigación en Biología Experimental, Facultad de Química, Mexico.
García-Soto
Jesús
J
Ongay-Larios
Laura
L
Coria
Roberto
R
Pedraza-Reyes
Mario
M
Arnau
José
J
Reyna-Lopez
Georgina
G
Martínez-Cadena
Guadalupe
G
D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D044385:GTP-Binding Protein alpha Subunits / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009089:Mucor / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D005656:Fungal Proteins; D044385:GTP-Binding Protein alpha Subunits; D011994:Recombinant Proteins