The LF1 gene of Chlamydomonas reinhardtii encodes a novel protein required for flagellar length control

Abstract

Flagellar length is tightly regulated in the biflagellate alga Chlamydomonas reinhardtii. Several genes required for control of flagellar length have been identified, including LF1, a gene required to assemble normal-length flagella. The lf1 mutation causes cells to assemble extra-long flagella and to regenerate flagella very slowly after amputation. Here we describe the positional cloning and molecular characterization of the LF1 gene using a bacterial artificial chromosome (BAC) library. LF1 encodes a protein of 804 amino acids with no obvious sequence homologs in other organisms. The single LF1 mutant allele is caused by a transversion that produces an amber stop at codon 87. Rescue of the lf1 phenotype upon transformation was obtained with clones containing the complete LF1 gene as well as clones that lack the last two exons of the gene, indicating that only the amino-terminal portion of the LF1 gene product (LF1p) is required for function. Although LF1 helps regulate flagellar length, the LF1p localizes almost exclusively in the cell body, with <1% of total cellular LF1p localizing to the flagella.

Figure 1.—

lf1 cells have defects in flagellar length control, assembly, and motility. (A) Histogram of flagellar lengths of wild-type (solid bars) and lf1 (open bars) cells. (B) DIC images of wild-type and lf1 cells. Bars, 10 μm. (C) Histogram of flagellar lengths of wild-type cells before deflagellation (solid bars). (D) Histogram of flagellar length of lf1 cells before deflagellation (solid bars), immediately following deflagellation (open bars), and 120 min after deflagellation (shaded bars). (E) Histogram of wild-type and lf1 cell swimming velocity.

Figure 2.—

Approximately 740-kb chromosome walk of linkage group II identifies location of LF1 gene. Contig of chromosome walk of linkage group II containing 17 of 101 BACs found in the region is shown. BAC names indicate plate number and well location. Molecular marker probes used to facilitate the walk are indicated on the top line. The walk spanned ∼740 kb and covered at least two genes identified by mutation, LF1 and PF12, separated by 3.5 MU. The entire contig is available at the Chlamydomonas Genetics Center web site: http://www.biology.duke.edu/chlamy_genome/BAC/GP366ext.html.

Figure 3.—

lf1 rescued cells rescue both the length defect and the regeneration defect of lf1 cells. (A) Histogram of flagellar lengths of wild-type cells (solid bars), lf1 cells (open bars), and lf1 cells rescued by transformation with the wild-type LF1 gene (shaded bars). DIC image of lf1 rescued cell is also shown. Bar, 10 μm. (B) Histogram of flagellar lengths of lf rescued cells before deflagellation (solid bars), immediately after deflagellation (open bars), and 120 min after deflagellation (shaded bars).

Figure 4.—

The LF1 gene contains eight exons and requires only the amino-terminal half of the protein for rescue of the lf1 phenotype. Shown is a restriction map of the 7.2-kb genomic plasmid (p7.2BB) containing the LF1 gene with the exons indicated as solid bars. Various subclones were able to rescue the lf1 phenotype upon transformation including three (p4.6BN, p3.9NN, and p5NN) that lack the 3′ end of the LF1 gene. Arrow indicates location of lf1 mutation.

Figure 5.—

LF1 encodes a novel protein of 804 amino acids. (A) Amino acid sequence of LF1. Underlined amino acid indicates location of lf1 amber stop codon. Sequence in boldface type is sufficient to rescue lf1 phenotype by transformation with p3.9NN plasmid (Figure 4). (B) Genomic sequence of the lf1 allele indicates a single-base-pair change from a G to a T leading to a premature stop codon at amino acid 87. (C) RNA blot analysis shows the 3.1-kb LF1 transcript is present in the lf1 mutant and (D) is not upregulated significantly after deflagellation. A total of 4–5 μg of poly(A) RNA was loaded per lane. P, predeflagellation. A cDNA fragment from the 3′ end of the LF1 gene was used as a probe. The same result was obtained with a probe of a cDNA fragment from the 5′ end of the LF1 gene (data not shown). A fragment of the CRY1 gene (encoding the ribosomal protein S14) was radiolabeled and used as a hybridization control for equal loading.

Figure 6.—

HA-tagged LF1 is present in the cell body and flagella. (A) Restriction map of 7.2-kb genomic plasmid showing the placement of the triple HA tag within the LF1 gene. (B) Immunoblot using an HA antibody shows four HA-tagged lf1 rescued strains (E1, G9, G12, and G12) express the HA-LF1 tagged protein, whereas an lf1 rescued strain transformed with a plasmid lacking the HA tag is not reactive to the HA antibody. (C) Immunoblot using an HA antibody indicates HA-LF1p is present in whole cells, cell bodies, and flagella. Lanes 1, 2, and 3 show protein from whole cell, cell body, and flagella (30 μg). Lanes 4 and 5 show protein from whole cells and cell bodies (2 × 10⁶). Lanes 6, 7, 8, 9, and 10 show protein from flagella isolated from 2 × 10⁶ (1×), 2 × 10⁷ (10×), 1 × 10⁸ (50×), 2 × 10⁸ (100×), and 2 × 10⁹ (1000×) cells. (D) Immunofluorescence of HA-LF1 tagged cells. Shown are phenotypically rescued strains, G2 and E1, stained with anti-HA antibody (1 and 3) and anti-tubulin antibody (2 and 4) and wild-type cells lacking the HA tag stained with anti-HA (5) and anti-tubulin (6) antibodies.