Structure/function of the human proton-coupled folate transporter (PCFT-SLC46A1)
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My thesis studies focused on four aspects of PCFT structure/function. (1) N-linked glycosylation: In vitro and in vivo enzymatic deglycosylation assays followed by Western blot analysis of HeLa cell membrane protein fractions transiently expressing PCFT suggested PCFT glycosylation. Single (N58Q, N68Q), or double (N58/68Q) PCFT mutants confirmed glycosylation but glycosylation was not required for function. Immunocytochemical studies indicated intracellular localization of the PCFT N- and C-termini. (2) Histidine residues: Ala substitution of all conserved His residues revealed functional changes only in H247A and H281A. The folic acid influx Kt was markedly decreased and there was a loss of substrate specificity for H247A-PCFT. Homology modeling predicted that H247 and S172 interact; these mutants were then shown to have similar functional phenotypes. Conversely, the folic acid influx Kt was markedly increased for H281A-PCFT. This was reversed, in part, by decreasing extracellular pH. H281A-PCFT transports protons when expressed in Xenopus oocytes. Hence, (i) H247 plays an important role in PCFT conformational equilibrium and substrate binding and (ii) H281A plays a role in proton binding to PCFT, which alters, allosterically, the affinity of PCFT for folate substrates, but is not required for proton translocation. (3) Charged amino acid residues: D156A, E232A, R148A, R376A, and E185A PCFT mutants lost function at pHe 5.5. Only E185A preserved function at pH 7.4. Most E185 mutants retained capacity for trans-stimulation at pH 5.5 and 7.4. The data suggested that E185 is required for proton coupling but, in its absence, the carrier is capable of function and exchange in the presence or absence of a proton-gradient. (4) Topology: Cysteineless PCFT constructs were fully functional even when Cys residues were inserted into predicted intracellular and extracellular loops. Using scanning cysteine accessibility (SCAM), the V141C- or T417C-Cysteineless-PCFT were extracellularly accessible to biotin-PEG2-maleimide, verifying elements of the PCFT secondary structure.
Unal, E. S. (2009). Structure/function of the human proton-coupled folate transporter (PCFT-SLC46A1) (Order No. 3356776). [Doctoral dissertation, Yeshiva University]. Available from ProQuest Dissertations & Theses Global. (305036892). ========Retrieved from https://ezproxy.yu.edu/login?url=https://www.proquest.com/dissertations-theses/structure-function-human-proton-coupled-folate/docview/305036892/se-2 Source: Dissertation Abstracts International, Volume: 70-04, Section: B, page: 2234.;Advisors: I. David Goldman.