我注意到了他反复说的加州大学的Dr. Milton Saier, Jr. 提到的“The best characterized members of the family are UhpT and GlpT, both of E. coli, for which detailed topological models have been presented (29, 90, 91).。我们参与争论的人，尽管不同意大阎的观点，但并不是不承认他的工作的重要和贡献。但是，再大的发现，也要沾边，不沾边，相差十万八千里的东西，人家根本就不会引用，道理不是很简单吗？
回复 'Fanreninus' 的评论 : 我是看此文没提C265。见下：Both G6PT and UhpT are Pi-linked antiporters [13,14]. Mutagenesis studies have shown that in UhpT, R46, R275, D388 and K391 are essential residues [15,16]. 他们不认为C265重要。不知道另有别人证实老阎93结果。 想了一个比喻。找通道就象走迷宫。66年一文指明了大方向（内外开口等）。老阎从内外开口走了他的迷宫，还说遇到过美女C265。（a residual in 93 文）。有几个后人走了老阎同一迷宫，没有见过C265。是不是老阎的结果不准？小颜则走了不同的迷宫，差不多走通了。还从迷宫中带出了宝贝（指导新药研发？）。
Huang Y, Lemieux MJ, Song J, Auer M, Wang DN. Structure and mechanism of the glycerol-3-
phosphate transporter from Escherichia coli. Science 2003;301:616–20. [PubMed: 12893936]
Published in final edited form as:
Mol Genet Metab. 2009 Jan; 96(1): 32–37.
Published online 2008 Nov 12. doi: 10.1016/j.ymgme.2008.10.00
Discussion
G6PT [9,14], GlpT [11], and UhpT [12] are members the organophosphate:Pi antiporter family of the major facilitator superfamily [10]. G6PT was shown to contain 10 transmembrane helices by protease protection and glycosylation scanning analysis [7]. However, recent homology modeling [19] based on the crystal structural of GlpT [17,18] predicts that G6PT contains 12 helices. Homology modeling [19] also proposes that amino acids essential for the activity of UhpT may play vital role in G6PT. In this study, we conducted structure-function studies of G6PT to determine if the predictions on which residues are critical for the activity of UhpT are pertinent to the structure of G6PT. We also re-examined the topology of G6PT. We show that structural requirements of G6PT and UhpT differ and that G6PT wild-type and glycosylation mutants exhibit similar sensitivity towards limited trypsin digestion. Taken together, the results support the 10 domain model of G6PT.
Both G6PT and UhpT are Pi-linked antiporters [13,14]. Mutagenesis studies have shown that in UhpT, R46, R275, D388 and K391 are essential residues [15,16]. R46 and R275 are proposed to be involved in substrate binding [15], and D388 and K391 in intrahelical salt bridge formation [16]. The corresponding residues in G6PT are R28, K240, H366, and V369. Homology modeling predicted that R28 and K240 in G6PT are 10.1 ? apart that could form part of the substrate-binding site [19]. We show that with the exception of R28 which is an essential residue in G6PT, K240, D388 and K391 are not, indicating that the structural requirements of G6PT and UhpT differed.
The main difference between the two topological models of G6PT is that residues 50 to 71, which constitute helix 2 in the 12-domain model [19], are situated in a 51-residue luminal loop 1 in the 10-domain model (Fig. 1). The glycosylation scanning study showed that the two G6PT mutants, T53N and S55N that generate potential glycosylation sites at N53SS and N55QS, respectively were utilized, which could only occur if the glycosylation sites were in the 51-residue luminal loop 1 in the 10-domain model [7] but not in helix-2 in the 12-domain model [19]. To explain the inconsistence to the 12-domain model G6PT, Almqvist et al [19] suggested that the two G6PT glycosylation mutants are probably misfolded in the ER membrane. Studies have shown that glycosylation of membrane proteins is critical for their membrane targeting and folding [27,28], and incorrect glycosylation can cause misfolding and preferential degradation of the misfolded proteins [29,30]. If the T53N and S55N mutants were misfolded in the ER membrane then they should exhibit differential stability and protease sensitivity as compared to the wild-type transporter. Our results show the contrary. First, the T53N and S55N mutants supported the synthesis of wild-type levels of G6PT protein in COS-1 cells, indicating that the mutations failed to destabilize mutant G6PT proteins. Secondly, limited trypsin digestion of wild-type G6PT produced an N-terminal polypeptide of 16-kDa and a C-terminal polypeptide of 22-kDa and limited trypsin digestion of G6PT T53N or S55N mutants produced an N-terminal polypeptide of 23-kDa and a C-terminal polypeptide of 22-kDa. However, in the presence of tunicamycin, limited trypsin digestion of G6PT S55N produced an N-terminal polypeptide of 16-kDa, indicating that the apparent difference in mobility between wild-type and mutant G6PT resulted from the added oligosaccharide side chains in the glycosylation mutants. Therefore, the introduced glycosylation site at either N53SS or N55QS in G6PT is utilized and the secondary structure of the glycosylated G6PT proteins is indistinguishable from the wild-type G6PT. Taken together, our data demonstrate that the glycosylation of G6PT does not cause G6PT to be misfolded in the ER membrane, again confirming the 10-transmembrane domain model of G6PT.
In summary, we demonstrate that the structural requirements of the two antiporters, G6PT and UhpT, are different and that G6PT is anchored in the ER by 10 transmembrane domains in contrast to the 12 domains used by UhpT.
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