Linked Life Data

9756872

Source:http://linkedlifedata.com/resource/pubmed/id/9756872

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
41
pubmed:dateCreated
1998-11-2
pubmed:abstractText
Hydropathy profile analysis of the amino acid sequence of the Na+/proline transporter of Escherichia coli (PutP) suggests that the protein consists of 12 transmembrane domains (TMs) which are connected by hydrophilic loops (Nakao, T., Yamato, I., and Anraku, Y. (1987) Mol. Gen. Genet. 208, 70-75). We have tested this prediction by applying a gene fusion approach in combination with a Cys accessibility analysis and site-specific proteolysis. Characterization of a series of PutP-alkaline phosphatase (PhoA) and PutP-beta-galactosidase (LacZ) hybrid proteins yields a reciprocal activity pattern of the reporter proteins that is in agreement with the topology of TMs III to XII of the 12-helix model. Placement of the PutP-PhoA and PutP-LacZ junction sites closer to the N terminus does not yield conclusive results. As a prerequisite for further topology studies, a functional PutP molecule devoid of all five native Cys residues (Cys-free PutP) is generated. Subsequently, amino acids in Cys-free PutP are replaced individually with Cys, and the accessibility of the sulfhydryl groups is analyzed. Surprisingly, Cys residues placed close to the N terminus of PutP (Ile-3 --> Cys, Thr-5 --> Cys) or into putative TM II (Ser-71 --> Cys, Glu-75 --> Cys) are highly accessible to membrane permeant and impermeant thiol reagents in intact cells. In contrast, Cys at the C terminus (Ser-502 --> Cys) reacts only with the membrane permeant but not with the impermeant reagent in intact cells. These results contradict the 12-helix motif and indicate a periplasmic location of the N terminus whereas the C terminus faces the cytoplasm. In addition, a transporter with Cys in place of Leu-37 (putative periplasmic loop (pL2) shows the same accessibility pattern as the Cys at the C terminus. Furthermore, PutP which has been purified and reconstituted into proteoliposomes in an inside-out orientation, is readily cleaved by the endoproteinase AspN before Asp-33 (pL2), Asp-112 (putative cytoplasmic loop (cL3), Asp-262 (cL7), and Asp-356 (cL9). These results suggest a cytosolic location of Asp-33 and Leu-37, thereby implying the formation of an additional TM formed by amino acids of pL2. Based on these observations, a new secondary structure model is proposed according to which the protein consists of 13 TMs with the N terminus on the outside and the C terminus facing the cytoplasm. The 13-helix structure is discussed as a common topological motif for all members of the Na+/solute cotransporter family.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
9
pubmed:volume
273
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
26400-7
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1998
pubmed:articleTitle
Topology of the Na+/proline transporter of Escherichia coli.
pubmed:affiliation
Universität Osnabrück, Fachbereich Biologie/Chemie, Arbeitsgruppe Mikrobiologie, Barbarastrabetae 11, D-49069 Osnabrück, Germany. jung_h@biologie.uni-osnabrueck.de
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't