23 January 2004. Going against the grain is never easy, and in science, especially, if you are proposing a rival model, you had better have strong evidence to support it. A paper appearing in this week’s early online PNAS rekindles the debate concerning the transmembrane topology of presenilin 1 (PS1). While it is generally accepted that PS1 has eight transmembrane domains (8-TM), this paper resurrects the hypothesis that PS1 assumes a 7-TM topology, and suggests it may have a role as a G-protein-coupled receptor (GPCR). The paper was contributed directly to the journal by the senior author.
Nazneen Dewji and S.J. Singer at the University of California, San Diego, have previously proposed a 7-TM model for PS1, (Dewji and Singer, 1997), which was not widely accepted in the field. In the present paper, these authors, with Dante Valdez, attempt to bolster their case by using more reliable monoclonal antibodies (mAb) in immunofluorescence-labeling studies and by transfecting double-null mouse embryonic stem (ES) cells with full-length human PS1. Additionally, they studied endogenous PS1 expression in human DAMI cells.
In the 8-TM model, the N-terminal, C-terminal, and loop region of PS1 all face the cytoplasm, as supported by previous studies using truncated PS1 fusion hybrids and intracellular immunostaining. However, these studies focused on PS1 pools in intracellular membranes, including the endoplasmic reticulum (ER) and Golgi membranes, while Dewji and colleagues studied the PS1 present at the cell surface. According to the 7-TM model, the PS1 N-terminal and loop domains are extracellular, while the C-terminal faces the cytoplasm.
Following prior work with polyclonal antibodies, the researchers in this study immunolabeled fixed, impermeable PS1 transfected and untransfected ES cells with monoclonal antibodies against the PS1 N-terminal domain and the PS1 loop domain. The immunolabeling showed cell-surface labeling of both the PS1 N-terminal and loop. The absence of fluorescence in transfected nonpermeabilized cells stained with an antibody against the PS1 C-terminal confirmed that the C-terminal was not accessible to the antibody at the cell surface and was located opposite from the N-terminal and the loop domains.
Dewji and colleagues also immunolabeled fixed, permeabilized transfected cells with PS1 loop mAb and C1 Ab, and observed cytoplasmic labeling in both cases. The latter result falls in line with the 7-TM proposed model, namely that the C-terminal of PS1 is on the cell interior.
To study endogenous PS1, the researchers immunolabeled untransfected human DAMI cells using the same antibodies against PS1 N-terminal and loop. In nonpermeabilized cells, PS1 N-terminal labeling was evident at the cell surface; upon permeabilization, however, the researchers saw some diffuse PS1 N-terminal and loop staining, indicating the presence of PS1 in intracellular membranes.
With these and some additional experiments on their side, the authors now write that “the evidence for the 8-TM topography is flawed,” partly because of the methods used, which include fusion proteins of truncated fragments of PS1. They assert that the PS1 N-terminal and loop domains are on the extracellular side of the membrane surface and the C-terminal is intracellular, making this model consistent with the functional prediction that PS1 has a role as a GPCR. The authors also argue that the “region of the C-terminal PS1 domain that binds the [brain protein] G0 shows significant local amino acid sequence homologies with the G-binding domains of the D2-dopaminergic and the 5-HT-1B receptors, both of which are 7-TM GPCRs.”
Even so, this 7-TM topology conflicts with other data that suggest the loop is a substrate for cytosolic enzymes, and has crucial binding sites for other proteins, according to an accompanying commentary by Jinoh Kim and Randy Schekman from the University of California, Berkeley. They note the possibility that PS1, like many other proteins, may exhibit more than one topological orientation. “Of course, two pools of PS1, one with an 8-TM topology retained within the cell and the other transported to the cell surface in a 7-TM orientation, would satisfy these seemingly contradictory observations,” the authors write.—Erene Mina.
Erene Mina is a graduate student at the University of California, Irvine.
Dewji NN, Valdez D, Singer SJ. The presenilins turned inside out: Implications for their structures and functions. PNAS 2004 Jan;101(4) 1057-1062.
Kim J, Schekman R. The ins and outs of presenilin 1 membrane topology. PNAS 2004 Jan;101(4) 905-906.