Once inserted, transmembrane segments of polytopic membrane proteins are generally considered stably oriented due to the large free energy barrier to topological reorientation of adjacent extramembrane domains. However, the topology and function of the polytopic membrane protein lactose permease of Escherichia coli are dependent on the membrane phospholipid composition, revealing topological dynamics of transmembrane domains after stable membrane insertion (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107-2116). In this study, we show that the high affinity phenylalanine permease PheP shares many similarities with lactose permease. PheP assembled in a mutant of E. coli lacking phosphatidylethanolamine (PE) exhibited significantly reduced active transport function and a complete inversion in topological orientation of the N terminus and adjoining transmembrane hairpin loop compared with PheP in a PE-containing strain. Introduction of PE following the assembly of PheP triggered a reorientation of the N terminus and adjacent hairpin to their native orientation associated with regain of wild-type transport function. The reversible orientation of these secondary transport proteins in response to a change in phospholipid composition might be a result of inherent conformational flexibility necessary for transport function or during protein assembly.Although considerable progress has been made in understanding the assembly of multispanning-membrane proteins (1, 2), the precise molecular events involved in the insertion, orientation, and proper formation of tertiary and quaternary structures of proteins in the membrane are not well defined. Most investigations have been focused on the role of amino acid sequence in directing the assembly of membrane proteins, whereas only a limited number of reports have addressed the effects of the native lipid environment in determining the correct insertion, folding, and topology of membrane proteins. Therefore, there is currently little understanding of, or ability to predict, how membrane protein topogenesis occurs in a given lipid environment. Whether there are constraints imposed on the topological organization of membrane proteins by phospholipid composition in addition to simply providing an amphipathic environment for maintenance of membrane protein conformation is also not clear.The most compelling evidence for a specific role for lipids in membrane protein topological organization is the requirement for phosphatidylethanolamine (PE) 1 for the proper orientation of the 12 transmembrane domains (TMs) of the lactose permease LacY of Escherichia coli (3). Assembly in the absence of PE results in a topological inversion of the N-terminal six TMs and their associated extramembrane domains. PE is required in a late step of maturation for the proper folding of the periplasmic extramembrane domain (P7) linking TMs VII and VIII (4). Proper folding of this domain is required for active (but not facilitated) transport of LacY substrates (5). The native topological org...

The H-2Db-restricted CD8 T cell immune response to influenza A is directed at two well-described epitopes, nucleoprotein 366 (NP366) and acid polymerase 224 (PA224). The responses to the two epitopes are very different. The epitope NP366-specific response is dominated by TCR clonotypes that are public (shared by most mice), whereas the epitope PA224-specific response is private (unique within each infected animal). In addition to being public, the NP366-specific response is dominated by a few clonotypes, when T cell clonotypes expressing the Vβ8.3 element are analyzed. Herein, we show that this response is similarly public when the NP366+Vβ4+ CD8 T cell response is analyzed. Furthermore, to determine whether these features resulted in differences in total TCR diversity in the NP366+ and PA224+ responses, we quantified the number of different CD8 T clonotypes responding to each epitope. We calculated that 50–550 clonotypes recognized each epitope in individual mice. Thus, although the character of the response to the two epitopes appeared to be different (private and diverse vs public and dominated by a few clonotypes), similar numbers of precursor cells responded to both epitopes and this number was of similar magnitude to that previously reported for other viral CD8 T cell epitopes. Therefore, even in CD8 T cell responses that appear to be oligoclonotypic, the total response is highly diverse.

The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium Escherichia coli. A topological model based on sequence analysis of the putative protein in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm had been proposed (J. Pi, P. J. Wookey, and A. J. Pittard, J. Bacteriol. 173:3622-3629, 1991). This topological model of PheP has been further examined by generating protein fusions with alkaline phosphatase. Twenty-five sandwich fusion proteins have been constructed by inserting the phoA gene at specific sites within the pheP gene. In general, the PhoA activities of the fusions support a PheP topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, alterations to the model, affecting spans III and VI, were indicated by this analysis and were supported by additional site-directed mutagenesis of some of the residues involved.The phenylalanine-specific permease (PheP) is a hydrophobic membrane protein of 458 amino acids that mediates the active transport of phenylalanine into Escherichia coli (26). It is highly homologous (60.4% identity) with the general aromatic amino acid permease (AroP) (14) and is a member of a superfamily of permeases, which are involved in the transport of amino acids in bacteria or yeasts (27,29). In addition, other protein sequences which have greater than 30% identity with PheP have been reported recently; the proteins include YTFD (hypothetical 51.7-kDa transport protein of E. coli (5), ANSP (L-asparagine permease of Salmonella typhimurium) (16), ROCE and ROCC (amino acid permeases of Bacillus subtilis) (11, 12), PROY (proline-specific permease of S. typhimurium) (18), LYP1 (lysine-specific permease of Saccharomyces cerevisiae) (34), ISP5 (putative amino acid permease of Schizosaccharomyces pombe) (32), INA1 (amino acid permease of Trichoderma harzianum) (37), VAL1 (valine-tyrosine-tryptophan amino acid permease of Saccharomyces cerevisiae) (33), and TAT2 (tryptophan permease of Saccharomyces cerevisiae) (33).Although phenylalanine uptake by PheP is driven by the proton motive force (24), the exact mechanism by which PheP mediates the active transport remains obscure. In this regard, information on the membrane topology of the protein is essential.The hydropathy plot of Engelman et al. (10) of the deduced amino acid sequence of PheP (26), together with the distribution of positively charged residues (23, 38) and the occurrence of the strong probability of ␤ turns (6), had allowed the prediction of a two-dimensional topological model for PheP, a polytopic integral membrane protein composed of 12 putative transmembrane domains (26) (Fig. 1).To test this model, we have used a genetic approach involving the construction of PheP-alkaline phosphatase fusions. Alkaline phosphatase requires export to the periplasm to form active enzyme. Thus in general, fusions of the alkaline phosphatase protein, without the leader sequence, to periplasmic domains of a membrane protein exhibit hig...

The phenylalanine-specific permease gene (pheP) of Escherichia coli has been cloned and sequenced. The gene was isolated on a 6-kb Sau3AI fragment from a chromosomal library, and its presence was verified by complementation of a mutant lacking the functional phenylalanine-specific permease. Subcloning from this fragment localized the pheP gene on a 2.7-kb HindIII-HindII fragment. The nucleotide sequence of this 2.7-kb region was determined. An open reading frame was identified which extends from a putative start point of translation (GTG at position 636) to a termination signal (TAA at position 2010). The assignment of the GTG as the initiation codon was verified by site-directed mutagenesis of the initiation codon and by introducing a chain termination mutation into the pheP-lacZ fusion construct. A single initiation site of transcription 30 bp upstream of the start point of translation was identified by the primer extension analysis. The pheP structural gene consists of 1,374 nucleotides specifying a protein of 458 amino acid residues. The PheP protein is very hydrophobic (71% nonpolar residues). A topological model predicted from the sequence analysis defines 12 transmembrane segments. This protein is highly homologous with the AroP (general aromatic transport) system of E. coli (59.6% identity) and to a lesser extent with the yeast permeases CAN1 (arginine), PUT4 (proline), and HIP1 (histidine) of Saccharomyces cerevisiae.

Site-directed mutagenesis has been used to identify a number of charged residues essential for the transport activity of the PheP protein. These residues are highly conserved in the cluster of amino acid transporters. However, some other conserved residues and a number of aromatic residues have been shown not to be essential for transport activity.