The @entity10043 - @entity5538 permease from @entity375 mediates the active transport through the plasma membrane of @entity5536 , @entity3625 , @entity2774 and @entity5538 using the proton electrochemical potential difference as an energy source. Analysis of the activity of strains mutated in a hydrophilic segment (371-377) of the polypeptidic chain has shown the involvement of this segment in the maintenance of the active three-dimensional structure of the carrier. In an attempt to identify permease domains that could interact functionally and/or physically with this segment, we looked for second-site mutations that could suppress the effects of @entity535 changes in this region. This paper describes a positive screen that has allowed the isolation of one suppressor from a permease mutant displaying the @entity69883 change (fcy2-20 allele), a substitution that induces a dramatic decrease in the affinity of the carrier for @entity5536 , @entity5538 and @entity3625 . The second-site mutation corresponds to the replacement of the @entity69884 . Its suppressive effect is shown to be a partial restoration of the binding of @entity5538 and @entity3625 to the permease. To test whether this second-site mutation is specific for the fcy2-20 allele, two double mutants were constructed (Fcy2p @entity69885 , @entity69884 and Fcy2p @entity69884 , @entity69886 ). Results obtained with these two double mutants showed that the suppressive effect of @entity69884 replacement was not specific for the original @entity69883 change. To understand the general effect of this @entity535 replacement for the three distinct double mutants, a strain overexpressing Fcy2p @entity69887 , was constructed. Kinetic analysis of this strain showed that, by itself, the @entity69884 change induced an improvement in the base-binding step that could account for its global suppressive effect. Moreover, @entity69884 induced a decrease in the turnover of the permease, thus showing the involvement of S272 in the translocation process. Taking into account the topological model of the permease proposed here, this Ser residue is probably located in a transmembrane amphipathic alpha-helix (TM5). The location and the observed decrease in the turnover of the carrier observed with the @entity69884 change lead us to propose that S272 could be part of a hydrophilic pore involved in the translocation of the base and/or the proton.

Screening of an intragenic second-site suppressor of @entity10043 - XXXX permease from @entity375 . Possible role of Ser272 in the base translocation process.

@entity3625 :: ('MESH:D019271', 'Chemical') :: ['hypoxanthine'] @entity10043 :: ('MESH:C030985', 'Chemical') :: ['purine'] @entity535 :: ('CHEBI:33704', 'Chemical') :: ['amino acid'] @entity69885 :: ('p|SUB|T|213|I', 'ProteinMutation') :: ['T213I'] @entity69883 :: ('p|SUB|N|374|I', 'ProteinMutation') :: ['N374I'] @entity2774 :: ('MESH:D006147', 'Chemical') :: ['guanine'] @entity5536 :: ('MESH:D000225', 'Chemical') :: ['adenine'] @entity69887 :: ('p|SUB|S|272|I', 'ProteinMutation') :: ['S272I'] @entity5538 :: ('MESH:D003596', 'Chemical') :: ['cytosine'] @entity69884 :: ('p|SUB|S|272|L', 'ProteinMutation') :: ['S272L', 'Ser272 residue by Leu', 'S272 L'] @entity375 :: ('4932', 'Species') :: ['Saccharomyces cerevisiae'] @entity69886 :: ('p|SUB|N|377|G', 'ProteinMutation') :: ['N377G']
@entity5538 :: (MESH:D003596,Chemical) :: ['cytosine']
