In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 98, No. 11 ( 2001-05-22), p. 6488-6493
Abstract:
The Arabidopsis thaliana AtHKT1 protein, a
Na + /K + transporter, is capable of mediating
inward Na + currents in Xenopus laevis oocytes and K + uptake in Escherichia coli .
HKT1 proteins are members of a superfamily of K + transporters. These proteins have been proposed to contain eight
transmembrane segments and four pore-forming regions arranged in a mode similar to that of a K + channel tetramer. However, computer
analysis of the AtHKT1 sequence identified eleven potential transmembrane segments. We have investigated the membrane topology of
AtHKT1 with three different techniques. First, a gene fusion alkaline phosphatase study in E. coli clearly defined the
topology of the N-terminal and middle region of AtHKT1, but the model for membrane folding of the C-terminal region had to be refined.
Second, with a reticulocyte-lysate supplemented with dog-pancreas microsomes, we demonstrated that N -glycosylation occurs
at position 429 of AtHKT1. An engineered unglycosylated protein variant, N429Q, mediated Na + currents in X.
laevis oocytes with the same characteristics as the wild-type
protein, indicating that N -glycosylation is not
essential for the functional expression and membrane targeting of AtHKT1. Five potential glycosylation sites were introduced into the
N429Q. Their pattern of glycosylation supported the model based on the E. coli -alkaline phosphatase data. Third,
immunocytochemical experiments with FLAG-tagged AtHKT1 in HEK293 cells revealed that the N and C termini of AtHKT1, and the regions containing
residues 135–142 and 377–384, face the cytosol, whereas the region of residues 55–62 is exposed to the outside. Taken together, our results
show that AtHKT1 contains eight transmembrane-spanning segments.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.101556598
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2001
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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