Plus and minus sexual agglutinins from Chlamydomonas reinhardtii

Abstract

Gametes of the unicellular green alga Chlamydomonas reinhardtii undergo sexual adhesion via enormous chimeric Hyp-rich glycoproteins (HRGPs), the plus and minus sexual agglutinins, that are displayed on their flagellar membrane surfaces. We have previously purified the agglutinins and analyzed their structural organization using electron microscopy. We report here the cloning and sequencing of the Sag1 and Sad1 genes that encode the two agglutinins and relate their derived amino acid sequences and predicted secondary structure to the morphology of the purified proteins. Both agglutinin proteins are organized into three distinct domains: a head, a shaft in a polyproline II configuration, and an N-terminal domain. The plus and minus heads are related in overall organization but poorly conserved in sequence except for two regions of predicted hydrophobic alpha-helix. The shafts contain numerous repeats of the PPSPX motif previously identified in Gp1, a cell wall HRGP. We propose that the head domains engage in autolectin associations with the distal termini of their own shafts and suggest ways that adhesion may involve head-head interactions, exolectin interactions between the heads and shafts of opposite type, and antiparallel shaft-shaft interactions mediated by carbohydrates displayed in polyproline II configurations.

Figure 1.

RNA Gel Blot Analysis of Wild-Type Sag1 and Sad1 Agglutinin Gene Expression. (A) and (B) Probes are shown in Figure 2. Sag1 (A) and Sad1 (B). wt, wild type; veg, vegetative (nongametic); gam, gametic; mid-1 and mid-2, mutants in the minus sex determination gene Mid; iso1 mt⁻, mutant that agglutinates both as plus and minus. Rehybridization of the blots with a probe made from the L27a 60S ribosomal protein gene served as a loading control. (C) and (D) RNA gel blot analysis of gametes carrying mutations in the Sag1 (C) and Sad1 (D) genes. The sag1-1 to sag1-5 mutants show varying levels of gene expression; the sag1-6 insertional mutant shows no signal. Sad1 expression is undetectable in the sad1-3 mutant and faint in sad1-1 and sad1-2. Loading levels are indicated by ethidium bromide (EtBr) staining of rRNA.

Figure 2.

Structure of the Sag1 and Sad1 Agglutinin Genes. The center of each diagram shows a restriction map (H, HindIII; X, XhoI; S, SalI). Above the map the agglutinin gene is shown; solid boxes represent coding sequences, open boxes 5′ and 3′ untranslated regions, and thin lines introns. A to D indicate four intron positions conserved in the two genes. The location of the deletion in the sad1-1 mutant is shown, as is the location of the insertion in the sag1-6 mutant. The position of the longest cDNA, the 5′ RACE product, and the RT-PCR products that were used to determine the mRNA structure are indicated below the restriction maps, as are sequences used as probes for Figure 1. The bottom of each diagram indicates which parts of the gene encode the N-terminal, shaft, and head domains (Figure 3).

Figure 3.

Predicted Amino Acid Sequences of the Sag1 and Sad1 Agglutinin Proteins. (A) Sag1. (B) Sad1. Numbered arrowheads indicate the locations of introns (the first intron in both sequences is in the 5′ untranslated region); putative N-glycosylation sites are underlined, and predicted signal sequences are boxed. N-terminal, shaft, and head domains are described in text. a.a., amino acids.

Figure 3.

Predicted Amino Acid Sequences of the Sag1 and Sad1 Agglutinin Proteins. (A) Sag1. (B) Sad1. Numbered arrowheads indicate the locations of introns (the first intron in both sequences is in the 5′ untranslated region); putative N-glycosylation sites are underlined, and predicted signal sequences are boxed. N-terminal, shaft, and head domains are described in text. a.a., amino acids.

Figure 4.

Morphology of Agglutinins and Related Cell Wall Proteins. (A) Plus agglutinin. Curved fibril protrudes beneath the large globular head. (B) Minus agglutinin. (C) Gp1 (upper) and Gp2 (lower) cell wall HRGPs. The kink in Gp1 is curved (left) or acute (right). (D) Plus agglutinins. Arrows indicate globular domains at the proximal shaft termini. Head 1 is globular; heads 2 and 3 are bilobed; head 4 displays a protruding curved fibril, shown at higher magnification in the inset. (E) Plus shaft showing head loop free of its globular head domain. (F) Diagram of the agglutinins. All images generated by QFDE-TEM. Bar = 50 nm.

Figure 5.

Far-UV CD Spectra of Plus and Minus Agglutinin at pH 7. Protein concentration was 0.1 mg/mL.

Figure 6.

Sequences of the Plus and Minus Agglutinins Heads Aligned by Computer Algorithms and Marked with Predicted Regions of Secondary Structure (See Icon Key at Top Left). Aligned amino acids are color coded (key at top left) to indicate their level of similarity. The αβ subdomains are shaded pink, followed by white P-rich subdomains, gray α1 subdomains, white long-unstructured subdomains, yellow α2 subdomains, and then white C-terminal subdomains. P, plus agglutinin heads; M, minus agglutinin heads.

Figure 7.

Subdomain Organization of the Plus and Minus Agglutinin Shafts. (A) Plus agglutinin shaft. (B) Minus agglutinin shaft. Repeating PPSPX motifs are in upper-case letters; PPX motifs are in upper-case letters in subdomains 2A and 2E. Nonrepeating residues and nonconforming positions in PPSPX units are in lower-case letters. Positions judged to be missing based on the nucleotide-level analysis of minus 2C are denoted with an asterisk. Block interruptions in subdomains 2A are indicated with angle brackets (< >); long non-PPSPX sequences in subdomains 2D are indicated with brackets. Numbered rows in the 2C domains indicate endoduplicated regions based on nucleotide alignments: two duplicated regions in plus and one region in minus.

Figure 7.

Subdomain Organization of the Plus and Minus Agglutinin Shafts. (A) Plus agglutinin shaft. (B) Minus agglutinin shaft. Repeating PPSPX motifs are in upper-case letters; PPX motifs are in upper-case letters in subdomains 2A and 2E. Nonrepeating residues and nonconforming positions in PPSPX units are in lower-case letters. Positions judged to be missing based on the nucleotide-level analysis of minus 2C are denoted with an asterisk. Block interruptions in subdomains 2A are indicated with angle brackets (< >); long non-PPSPX sequences in subdomains 2D are indicated with brackets. Numbered rows in the 2C domains indicate endoduplicated regions based on nucleotide alignments: two duplicated regions in plus and one region in minus.

Figure 8.

Sequences of the Plus and Minus Agglutinin N-Terminal Domains Aligned by Computer Algorithms and Marked with Predicted Regions of Secondary Structure (See Icon Key at Top Left). Aligned amino acids are color coded (key at top left) to indicate their level of similarity. P, plus agglutinin N-terminal domains; M, minus agglutinin N-terminal domains.