Subcellular localization of galactose-1-phosphate uridylyltransferase in the yeast Saccharomyces cerevisiae.
The enzyme galactose-1-phosphate uridylyltransferase (GALT) catalyzes the second step of the Leloir pathway of galactose metabolism, following galactokinase (GALK) and preceding UDP-galactose-4-epimerase (GALE). Impairment of GALT in humans results in the potentially lethal disorder classic galactosemia. Standard lysis protocols of bacteria, yeast, or mammalian cells release all three Leloir enzymes in the soluble fraction, leading to the historical assumption that all three function as free cytosolic enzymes. We have tested this assumption with regard to GALT in vivo using the yeast Saccharomyces cerevisiae, by linking a GFP-tag onto the amino terminus of Gal7p, the endogenous yeast GALT. We find clear evidence of localization of the fusion protein to discrete spots in the cytoplasm of the majority of cells expressing all three Leloir enzymes, although GFP alone appears freely cytosolic. In contrast, yeast expressing GFP-Gal7p but lacking Gal1p (GALK), Gal10p (GALE), or both do not demonstrate spots in the majority of cells, implicating a role, either direct or indirect, for these other Leloir proteins in the Gal7p localization process. Preliminary truncation experiments reveal that amino acids 1-134 of Gal7p are sufficient to drive localization of the fusion protein, while amino acids 1-66 are not. Finally, GFP-tagged human GALT expressed in yeast also localizes to spots, demonstrating that at least some of the intrinsic determinants of localization have been conserved. These observations raise the intriguing possibility that GALT may function in a sequestered rather than a freely diffusible state, and that this subcellular organization may have been conserved through evolution.
Christacos
N C
NC
Graduate Program in Genetics and Molecular Biology, Department of Genetics, Graduate Program in Biochemistry and Molecular Biology, Emory University, 1462 Clifton Road NE, Atlanta, Georgia 30322, USA.
Marson
M J
MJ
Wells
L
L
Riehman
K
K
Fridovich-Keil
J L
JL
D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology*; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000378:metabolism*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase