Homocitrate synthase is located in the nucleus in the yeast Saccharomyces cerevisiae.
We have generated monoclonal antibodies against nuclear proteins from the yeast Saccharomyces cerevisiae. The monoclonal antibodies react with proteins of 47 and 49 kDa on immunoblots and with partially overlapping sets of proteins on two-dimensional nonequilibrium pH gradient electrophoresis-SDS blots. Immunofluorescence localization shows a nuclear staining pattern. Immunoscreening a yeast expression library yielded five independent full-length clones of two open reading frames from chromosome IV, corresponding to YDL182w (LYS20) and YDL131w in the Saccharomyces genome data base. These two open reading frames are predicted to encode homocitrate synthase isozymes of 47 and 49 kDa, respectively. A clone carrying YDL182w was sequenced in its entirety and directs the expression of a 47-kDa protein in Escherichia coli. A clone carrying YDL131w expresses a 49-kDa protein in E. coli. Yeast grown in minimal medium plus lysine show significant reductions in nuclear immunofluorescence staining. Cell fractionation studies localize the 47- and 49-kDa proteins to the nucleus. Nuclear fractionation studies reveal that a portion of the 47- and 49-kDa proteins can only be extracted with DNase digestion and high salt. The localization of homocitrate synthase to the nucleus is unexpected given previous reports that homocitrate synthase is present in mitochondria and the cytoplasm in S. cerevisiae.
Chen
S
S
Department of Anatomy and Cell Biology, Health Science Center, University of Florida, Gainesville, Florida 32610, USA.
Brockenbrough
J S
JS
Dove
J E
JE
Aris
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002467:Cell Nucleus / Q000201:enzymology*; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015698:Genomic Library; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000032:analysis; D007652:Oxo-Acid-Lyases / Q000032:analysis* / Q000096:biosynthesis* / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C021365:homocitrate synthase; D007652:Oxo-Acid-Lyases