Fragmentation of dihydroxyacetone kinase 1 from Saccharomyces cerevisiae indicates a two-domain structure.
Global protein expression in Saccharomyces cerevisiae strains either deleted for both yeast dihydroxyacetone kinases (DAK1 and DAK2) or overexpressing DAK1, was characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). We found protein expression in the double deletion strain to be highly similar to wild-type. In the strain overexpressing Dak1p, nine spots representing fragments of the Dak1p protein in the size range 40-20 kDa and amounting to approximately 30% of total Dak1p, were discovered (native size Dak1p migrates at roughly 60 kDa). Fragments were characterized by matrix-assisted laser desorption/ionization mass spectrometry and electrospray mass spectrometry analyses to represent either the N- or the C-terminal part of the DAK1 protein. Cleavage points, predicted from mass spectrometry and 2-D PAGE data, mapped almost exclusively in the middle region showing low sequence conservation between Dak1p and its closest homologues. We hypothesize that observed Dak1p fragments represent stable structural domains shielded from access by native endoproteases. Furthermore, overexpressing Dak1p with the non-native N-terminus (M)A-, resulted in native size Dak1p and N-terminal Dak1p fragments appearing in two major 2-D PAGE forms of approximately equal size and abundance, but with slightly different isoelectric points. However, when overexpressing Dak1p with the native N-terminus (M)S-, only the more acidic 2-D PAGE form appeared. In the N-terminal acetyltransferase mutant nat1delta, (M)A-Dak1p species were converted into the basic form, arguing twin spots to represent forms with acetylated and deacetylated N-termini. Data thus indicated that (M)A-N-termini, in the Dak1p context, were NatA substrates recognized with 50% lower efficiency than (M)S-N-termini.
Molin
Mikael
M
Department of Cell and Molecular Biology--Microbiology, Göteborg University, Göteborg, Sweden. mikael.molin@gmm.gu.se
Larsson
Thomas
T
Karlsson
Karl-Anders
KA
Blomberg
Anders
A
D000107:Acetylation; D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D004271:DNA, Fungal; D010446:Peptide Fragments; D017853:Phosphotransferases (Alcohol Group Acceptor); C023132:glycerone kinase