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. 2001 Mar;12(3):739-51.
doi: 10.1091/mbc.12.3.739.

The Chlamydomonas PF6 Locus Encodes a Large Alanine/Proline-Rich Polypeptide That Is Required for Assembly of a Central Pair Projection and Regulates Flagellar Motility

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The Chlamydomonas PF6 Locus Encodes a Large Alanine/Proline-Rich Polypeptide That Is Required for Assembly of a Central Pair Projection and Regulates Flagellar Motility

G Rupp et al. Mol Biol Cell. .
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Abstract

Efficient motility of the eukaryotic flagellum requires precise temporal and spatial control of its constituent dynein motors. The central pair and its associated structures have been implicated as important members of a signal transduction cascade that ultimately regulates dynein arm activity. To identify central pair components involved in this process, we characterized a Chlamydomonas motility mutant (pf6-2) obtained by insertional mutagenesis. pf6-2 flagella twitch ineffectively and lack the 1a projection on the C1 microtubule of the central pair. Transformation with constructs containing a full-length, wild-type copy of the PF6 gene rescues the functional, structural, and biochemical defects associated with the pf6 mutation. Sequence analysis indicates that the PF6 gene encodes a large polypeptide that contains numerous alanine-rich, proline-rich, and basic domains and has limited homology to an expressed sequence tag derived from a human testis cDNA library. Biochemical analysis of an epitope-tagged PF6 construct demonstrates that the PF6 polypeptide is an axonemal component that cosediments at 12.6S with several other polypeptides. The PF6 protein appears to be an essential component required for assembly of some of these polypeptides into the C1-1a projection.

Figures

Figure 1
Figure 1
Central pair structure. (Left) Diagrammatic representation of the multiple projections associated with the central pair apparatus; redrawn from Mitchell and Sale (1999). The C1 microtubule (left) is associated with two 18-nm-long projections (1a and 1b) and two smaller projections (1c and 1d). Projections 1b and 1d appear to be linked by a thin sheath. The C2 microtubule (right) is associated with two 8-nm-long projections (2a and 2b) and one smaller projection (2c). The C1 and C2 microtubules appear to be linked by a two-membered bridge and a diagonal link that extends from the C2 microtubule to the C1 microtubule near the base of the C1–1b projection. (Middle) Image average of central pair structure from wild-type (wt) axonemes (n = 32). (Right) Image average of central pair structure from pf6-2 axonemes (n = 27). Note the absence of the 1a projection on the C1 microtubule.
Figure 2
Figure 2
Electron microscopic analysis of axonemes from wild-type (wt) and mutant Chlamydomonas cells. (A) Transverse sections of flagellar axonemes reveal that the C1-1a projection present in wild-type samples is missing from pf6-2 and pf6-1 preparations (arrows). (B) Longitudinal images reveal two rows of projections repeating at precise 16-nm intervals in wild-type samples, but one row of projections is missing in the pf6–2 axonemes (brackets). The observed central pair microtubule is identified as C1 based on the length of the remaining associated projections. (C) Rescue of the pf6 mutant motility defect by transformation with a clone containing a full-length wild-type copy of the PF6 gene is also accompanied by restoration of the C1-1a projection in both pf6-2 and pf6-1, as observed in axoneme cross-sections (arrows). Bars, 25 nm.
Figure 3
Figure 3
Recovery of the PF6 transcription unit. (A) Partial restriction maps of the genomic DNA region containing the PF6 transcription unit from wild-type (wt) and pf6-2. Also indicated on the diagram is the location of the NIT1 plasmid insertion in pf6-2 and the position of the flanking clone FC-1 (black box) representing the PvuII restriction fragment recovered from a size-fractionated mini-library. S, SacI sites; N, NotI sites. (B) Selected SacI fragments (A–E) that were used to map the boundaries of the plasmid insertion in pf6-2 and determine the size of the PF6 transcription unit. (C) Alignment of the six overlapping phage clones recovered with the flanking clone FC-1 (black boxes) and the three plasmid clones, derived from the λL1a insert, that contain the PF6 transcription unit. The plasmid pSET-41 contains an epitope-tagged PF6 gene construct. Also indicated are the number of rescued strains observed by cotransformation with the selected clones.
Figure 4
Figure 4
Expression of the PF6 transcript. (A) Northern blot loaded with total RNA isolated from wild-type, pf6 mutants, and a rescued pf6 strain before (0) and forty-five (45) min after deflagellation. This blot was probed with an RT-PCR product from within probe C (Figure 3) and is representative of blots hybridized with probes B, C, and D. The ∼7-kb PF6 transcript is indicated (arrow). The slight upward shift in transcript migration in the pf6-2 rescue lanes appears to result from differences in the loading of these lanes relative to the others. (B) The same Northern blot shown above was rehybridized with a probe for the CRY1 gene, which encodes the ribosomal S14 protein subunit (Nelson et al., 1994), as a control for loading of the RNA samples.
Figure 5
Figure 5
Structure of the PF6 gene and polypeptide. (A) Diagram of the proposed intron-exon structure of the PF6 transcription unit. Open rectangles indicate exons and solid lines indicate introns. The predicted translation start (ATG) and stop sites are indicated, as well as the putative polyadenylation signals (Poly-A). The location of the epitope tag inserted into intron 8 is marked. (B) Diagrammatic representation of the domain structure of the PF6 gene product. Indicated are the location of proline-rich (P; cross-hatched boxes), alanine-rich (A; single-hatched boxes), basic (B; gray, shaded boxes), and probable coiled-coil (c-c; black boxes) domains and the location of the inserted epitope tag. a.a., amino acid.
Figure 6
Figure 6
Predicted amino acid sequence of the PF6 gene product (GenBank accession AF327876).
Figure 7
Figure 7
Clustal W alignment of the PF6 gene product and the partial EST derived from a human testis cDNA library. The alignment in this region shows 41% similarity and 23% identity between the two sequences.
Figure 8
Figure 8
Localization of the PF6 gene product. (A) Whole axonemes isolated from wild-type (wt), pf6-1, pf6-2, and a pf6-2 strain rescued with the epitope-tagged PF6 gene (pf6–2 ET) were separated on 6% acrylamide gels, blotted onto polyvinylidene difluoride, and stained with a reversible total protein stain (left) before immunolabeling with an antibody directed against the nine-amino acid HA epitope (right). The HA antibody recognized a single polypeptide present only in the pf6-2 ET sample that migrates slightly larger than 250 kDa in this gel system (arrow). (B) Indirect immunofluorescent localization of the PF6 gene product. pf6-2 (a–c) and pf6-2 ET (d–f) cells were labeled with antibodies against either α-tubulin (a and d) or the HA-epitope (b, e, and f). All of the recorded cells had flagella similar to those depicted in a and d. The tagged PF6 polypeptide is present along the entire length of the axoneme in pf6-2 ET cells (e and f). Control samples labeled with secondary antibody alone (c) demonstrate background cell body autofluorescence.
Figure 9
Figure 9
Polypeptides associated with the PF6 gene product. (A) Isolated flagella from an epitope-tagged, pf6-2 rescued strain were subjected to successive extractions and then analyzed on a Western blot probed with the anti-HA antibody. Lane 1, membrane plus matrix fraction; lane 2, whole axonemes; lane 3, 0.6 M NaCl supernatant; lane 4, 0.6 M NaCl pellet; lane 5, 0.2 M KI supernatant; lane 6, 0.2 M KI pellet. Note that only a portion of the epitope-tagged PF6 protein is solubilized by 0.6 M NaCl extraction (lane 3), whereas all of the remaining PF6 protein is released after treatment with 0.2 M KI (lane 5). (B) The 0.2 M KI extracts were subsequently fractionated by sucrose density gradients. The HA-tagged PF6 protein sedimented at ∼12.6S, peaking in fraction 9. Silver-stained gels indicated that the polypeptide composition of these fractions was still quite crude, but several polypeptides observed in the rescued sample (pf6-2r) appear to be missing in the comparable fraction from the pf6-2 gradient. The position of molecular weight standards is indicated. The arrow on the right indicates the approximate position of the HA-tagged PF6 polypeptide seen on Western blots.

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