Abstract
A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), is found in a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane. This motif is located within the cytoplasmic side of transmembrane domain 2 (TM-2) and extends through the hydrophilic loop that connects transmembrane domains 2 and 3. The motif is repeated again in the second half of the protein. In a previous study concerning the loop 2/3 motif (Jessen-Marshall, A. E., Paul, N. J., and Brooker, R. J. (1995) J. Biol. Chem. 270, 16251-16257), it was shown that the conserved aspartate at the fifth position in the motif is critical for transport activity since a variety of site-directed mutations were found to greatly diminish the rate of transport. In the current study, two of these mutations, in which the conserved aspartate was changed to threonine or serine, were used as parental strains to isolate second site suppressor mutations that restore transport function. A total of 10 different second site mutations were identified among a screen of 19 independent mutants. One of the suppressors was found within loop 1/2 in which Thr-45 was changed to arginine. Since the conserved aspartate and position 45 are at opposite ends of TM-2, these results suggest that the role of the conserved aspartate residue in loop 2/3 is to influence the topology of TM-2. Surprisingly, the majority of suppressor mutations were found in the second half of the permease. All of these are expected to alter helix topology in either of two ways. Some of the mutations involved residues within transmembrane segments 7 and 11 that produced substantial changes in side chain volume: TM-7 (Cys-234-->Trp or Phe, Gln-241-->Leu, and Phe-247-->Val) and TM-11 (Ser-366-->Phe). Alternatively, other mutations were highly disruptive substitutions at the ends of transmembrane segments or within hydrophilic loops (Gly-257-->Asp, Val-367-->Glu, Ala-369-->Pro, and a 5-codon insertion into loop 11/12). It is hypothesized that the effects of these suppressor mutations are to alter the helical topologies in the second half of the protein to facilitate a better interaction with the first half. Overall, these results are consistent with a transport model in which TM-2 acts as an important interface between the two halves of the lactose permease. According to our tertiary model, this interaction occurs between TM-2 and TM-11.
Highlights
The uptake of a variety of solutes, including sugars, amino acids, and inorganic ions, is mediated by integral membrane proteins known as cation/substrate cotransporters or symporters
Isolation of Second Site Suppressor Mutants—In our previous work, it was shown that the aspartate residue at position 68 in the lactose permease is important for transport activity [13]
The results of the current study provide evidence that the aspartate within the GXXX(D/E)(R/K)XG(R/K)(R/K) motif is important in maintaining the conformation of the permease
Summary
The uptake of a variety of solutes, including sugars, amino acids, and inorganic ions, is mediated by integral membrane proteins known as cation/substrate cotransporters or symporters. The lactose permease of Escherichia coli has provided a model system in which to investigate the molecular mechanism of symport [1, 2] This protein is found in the E. coli cytoplasmic membrane and couples the transport of Hϩ and lactose. Evolutionary analyses have indicated that the lactose permease is a member of a large group of solute transporters that are homologous to each other (8 –10) This superfamily includes proteins that transport substrates such as sugars, Kreb’s cycle intermediates, and antibiotics. Secondary structural models for members of the USA superfamily are commonly found to predict a membrane protein with 12 transmembrane segments traversing the membrane in an ␣-helical manner. Based on the structural characteristics of the members of the USA superfamily, we have recently proposed a three-dimensional model that describes the orientation of the 12 transmembrane segments [11]. The serine and threonine mutants were used as parental strains to identify second site suppressor mutations that restore transport function
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
