APH-1 is a multipass membrane protein essential for the Notch signaling pathway in Caenorhabditis elegans embryos

Abstract

Early embryonic cells in Caenorhabditis elegans embryos interact through a signaling pathway closely related to the Notch signaling pathway in Drosophila and vertebrates. Components of this pathway include a ligand, receptor, the presenilin proteins, and a novel protein, APH-2, that is related to the Nicastrin protein in humans. Here we identify the aph-1 gene as a new component of the Notch pathway in Caenorhabditis elegans. aph-1 is predicted to encode a novel, highly conserved multipass membrane protein. We show that aph-1 and the presenilin genes share a similar function in that they are both required for proper cell-surface localization of APH-2/Nicastrin.

Figure 1

aph-1 and wild-type embryos. (a) Living wild-type embryo viewed by light microscopy; the arrow indicates the anterior half of the pharynx and the arrowhead indicates the posterior half. (b) Immunostaining of pharyngeal cells; markers as in a. (c) Hypodermal cell boundaries delineated by green fluorescent protein expression; 10 cells are visible in the lateral row. (d) Four-cell embryo showing expression of the ligand APX-1. APX-1 is visible in the posterior-most cell (P2; white arrowhead) as a line contacting one of the GLP-1-expressing cells (ABp; black arrowhead); embryos oriented with the anterior to the left. (e–h) aph-1 mutant embryos staged, prepared, and labeled as above. Note the extra hypodermal cells at the first, second, and fourth positions in g. e and g are aph-1(zu147), and f and h are aph-1(zu123).

Figure 2

(a) alignment of the predicted APH-1 protein of C. elegans with related proteins in Drosophila (33% identity, GenBank accession no. AAF51212) and humans (33% and 34% identity, GenBank accession nos. AAD34072 and AL136671, respectively). Each of the two human genes also has a highly related mouse gene, which is not shown here (GenBank accession nos. AC015932.1 and AK002310). Identical amino acids are black and conserved amino acids are gray; predicted transmembrane domains are overlined. Asterisks correspond to positions of aph-1 mutations as follows. zu123, Met to Ile; or28, Gly to Asp; zu147, Arg to opal stop codon. (b) APH-1 hydrophobicity plots (21); alignments as shown in a; predicted membrane-spanning regions are numbered above the plots.

Figure 3

GLP-1 and APH-2 localization in 4-cell stage embryos. The signaling cell (P2; white arrowhead) and responding cell (ABp; black arrowhead) are indicated in all panels. (a) Immunostaining of GLP-1 in wild type. GLP-1 is expressed on the surface of the anterior-most cell (Left) and its sister (black arrowhead). (b) Immunostaining of APH-2 in wild type. APH-2 is visible on the surface membranes of all four cells. (c and d) aph-1(zu123) embryos labeled as above. (e and f) Embryos deficient in presenilins [sel-12(ty11); hop-1(RNAi)] labeled as above. The level of APH-2 immunostaining is highest when cells are mid-interphase; in the images shown, the GLP-1-expressing cells appear to express a higher level of APH-2 because they are slightly more advanced in the cell cycle than the other two cells.