Additional evidence for an eight-transmembrane-domain topology for Caenorhabditis elegans and human presenilins

Abstract

Presenilins have been implicated in the genesis of Alzheimer's disease and in facilitating LIN-12/Notch activity during development. All presenilins have multiple hydrophobic regions that could theoretically span a membrane, and a description of the membrane topology is a crucial step toward deducing the mechanism of presenilin function. Previously, we proposed an eight-transmembrane-domain model for presenilin, based on studies of the Caenorhabditis elegans SEL-12 presenilin. Here, we describe experiments that support the view that two of the hydrophobic regions of SEL-12 function as the seventh and eighth transmembrane domains. Furthermore, we have shown that human presenilin 1 behaves like SEL-12 presenilin when analyzed by our methods. Our results provide additional experimental support for the eight-transmembrane-domain model of presenilin topology.

Figure 1

Hydrophobicity plot of SEL-12 and schematic representation of SEL-12 constructs. (A) Hydrophobicity plot of SEL-12 constructed by using the Kyte–Doolittle algorithm (25) with a window size of 15. HRs are numbered as in ref. . (B) SEL-12 constructs used in the LacZ fusion approach and the rescue assay. SEL-12ΔHR6–8∷LacZ, LacZ placed after the HR9 of SEL-12 with the region of HR6 through HR8 deleted; SEL-12ΔHR6–8∷TM∷LacZ, a construct similar to SEL-12ΔHR6–8∷LacZ but including a synthetic transmembrane domain (21) at the fusion junction (amino acid P410 of SEL-12) between sel-12 and lacZ cDNAs.

Figure 2

Rescue of the sel-12(ar171) Egl and abnormal vulva phenotype by the SEL-12 constructs. The data shown for transgenic lines are generated by injecting a construct that places a modified sel-12 cDNA (encoding SEL-12HR8*, in which the hydrophobicity of HR8 has been increased; see Fig. 3) or a truncated sel-12 cDNA (encoding SEL-12ΔHR8 or SEL-12ΔHR8–9) under the control of sel-12 5′ flanking sequence. Each line in the histogram represents data for an independent transgenic line and the number of hermaphrodites scored is shown above each line. The altered protein encoded by the transgene is indicated on the horizontal axis. The percentage of rescued hermaphrodites is indicated on the vertical axis. Transgenic animals expressing the pLSX vector do not exhibit any rescuing activity (4).

Figure 3

Hydrophobicity plots of SEL-12 and SEL-12HR8*. The algorithm of Kyte and Doolittle (25) was used with a window size of 15 residues. (A) Hydrophobicity plot of SEL-12. (B) Hydrophobicity plot of SEL-12HR8*, a modified SEL-12 protein, in which the six amino acids of HR8 (KASSYF, amino acids 374–379) are replaced by the seven amino acids VAVVLAP. This modification makes the new HR8 (indicated by * in the hydrophobicity plot) of the modified SEL-12 protein highly hydrophobic. The arrow indicates the fusion point of SEL-12HR8*∷LacZ and SEL-12HR8*∷TM∷LacZ at the newly introduced L378.

Figure 4

Hydrophobicity plot of human PS1 and schematic representations of PS1∷LacZ and PS1∷TM∷LacZ constructs. (A) Hydrophobicity plot of PS1, using a window size of 15 (ref. 25). The hydrophobic regions are numbered as in ref. . (B) PS1 constructs used in the LacZ fusion approach for topological studies. PS1HR7∷LacZ, LacZ placed after the HR7 of PS1; PS1HR7∷TM∷LacZ, a fusion similar to PS1HR7∷LacZ with a synthetic transmembrane domain at the fusion junction (amino acid L377 of PS1); PS1HR9∷LacZ, LacZ placed after the HR9 of PS1; PS1HR9∷TM∷LacZ, a fusion similar to PS1HR9∷LacZ with a synthetic transmembrane at the fusion junction (amino acid P429 of PS1); PS1ΔHR8∷LacZ, LacZ placed after the HR9 of PS1 with the HR8 deleted; PS1ΔHR8∷TM∷LacZ, a fusion similar to PS1ΔHR8∷LacZ with a synthetic transmembrane domain at the fusion junction (amino acid P429 of PS1).

Figure 5

Inferred topology of presenilin proteins. Arabic numerals indicate the transmembrane domains deduced from this study of SEL-12 and PS1 and our previous study of SEL-12 (13). Horizontal rectangles indicate the seventh or the tenth hydrophobic region in presenilins, which do not appear to span the membrane. Asterisks indicate the positions of mutations found in PS1 that are associated with early-onset familial Alzheimer’s disease (ref. and references therein).