MUTATIONAL ANALYSIS OF THE SNF3 GLUCOSE TRANSPORTER OF SACCHAROMYCES-CEREVISIAE

被引:65
作者
MARSHALLCARLSON, L
CELENZA, JL
LAURENT, BC
CARLSON, M
机构
[1] COLUMBIA UNIV COLL PHYS & SURG,DEPT GENET & DEV,NEW YORK,NY 10032
[2] COLUMBIA UNIV COLL PHYS & SURG,INST CANC RES,NEW YORK,NY 10032
关键词
D O I
10.1128/MCB.10.3.1105
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The SNF3 gene of Saccharomyces cerevisiae encodes a high-affinity glucose transporter that is homologous to mammalian glucose transporters. Point mutations affecting the function of the transporter were recovered from the genomes of four snf3 mutants and characterized. Two of the mutations introduced a charged amino acid into the first and second predicted membrane-spanning regions, respectively. The analogs of a bifunctional SNF3-lacZ fusion containing these two mutations were constructed, and the mutant fusion proteins were not localized to the plasma membrane, as judged by immunofluorescence microscopy. The third mutation produced a valine-to-isoleucine substitution in hydrophobic region 8, and the corresponding mutant fusion protein was correctly localized. The finding that this conservative change causes a transport defect is consistent with the possibility that this transmembrane region, which could exist as an amphipathic α-helix, forms part of the glucose channel through the membrane. The fourth snf3 allele harbored an ochre mutation midway through the coding sequence. We have also constructed mutations in the cloned SNF3 gene. A major difference between the yeast SNF3 protein and mammalian glucose transporters is the presence in the SNF3 protein of an additional 303 amino acids at the C. terminus. Analysis of a series of C-terminal deletions and fusions to lacZ showed that this C-terminal region is important, but not essential, for transport function. We also report the genetic mapping of the SNF3 locus on the left arm of chromosome IV.
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页码:1105 / 1115
页数:11
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