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. 2005 Oct;16(10):4509-18.
doi: 10.1091/mbc.e05-04-0347. Epub 2005 Jul 19.

ATP Production in Chlamydomonas Reinhardtii Flagella by Glycolytic Enzymes

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Free PMC article

ATP Production in Chlamydomonas Reinhardtii Flagella by Glycolytic Enzymes

Beth F Mitchell et al. Mol Biol Cell. .
Free PMC article

Abstract

Eukaryotic cilia and flagella are long, thin organelles, and diffusion from the cytoplasm may not be able to support the high ATP concentrations needed for dynein motor activity. We discovered enzyme activities in the Chlamydomonas reinhardtii flagellum that catalyze three steps of the lower half of glycolysis (phosphoglycerate mutase, enolase, and pyruvate kinase). These enzymes can generate one ATP molecule for every substrate molecule consumed. Flagellar fractionation shows that enolase is at least partially associated with the axoneme, whereas phosphoglycerate mutase and pyruvate kinase primarily reside in the detergent-soluble (membrane + matrix) compartments. We further show that axonemal enolase is a subunit of the CPC1 central pair complex and that reduced flagellar enolase levels in the cpc1 mutant correlate with the reduced flagellar ATP concentrations and reduced in vivo beat frequencies reported previously in the cpc1 strain. We conclude that in situ ATP synthesis throughout the flagellar compartment is essential for normal flagellar motility.

Figures

Figure 1.
Figure 1.
Electron micrographs illustrating the restricted pathway for diffusion of ATP from the cell body into the flagellar compartment. (A) Thin section through a Chlamydomonas cell body and one of the two flagella. Boxed region in A is enlarged in B, which shows that ATP synthesized by mitochondria (mito) must pass the basal body (bb) to the flagellum (fla) through a transition zone that links flagellar microtubules to the cell membrane (arrow). Bar, (A) 2 μm, (B) 100 nm.
Figure 2.
Figure 2.
Identification of CPC1 complex subunits. CPC1 extracted from wild-type axonemes by 0.2 M KI cosediments on a sucrose gradient with four other proteins. (A) Stained gel of sucrose gradient fractions from wild-type KI extracts with CPC1 complex subunits (arrows) labeled by apparent size. The 265-kDa band was previously determined to be CPC1 (Zhang and Mitchell, 2004). (B) Domain structure of CPC1 and the two novel subunits identified by proteomic analysis (see text and Table 1). The 350-kDa protein contains domains with homology to calpain (C), adenylate kinase (AK), and guanylate kinase (GK); CPC1 contains one AK domain; the 135-kDa protein contains two armadillo repeat domains (AR). (C) Immunoblots of the sucrose gradient fractions shown in A were probed with the indicated antibodies and reveal the cosedimentation of CPC1, HSP70A, and enolase (fractions 5 and 6). Additional peaks of HSP70A and enolase occur higher in the gradient. (D) Gel and corresponding immunoblots of wild-type (WT), central pairless (pf18), and cpc1 axonemal proteins probed with antibodies to HSP70A (HSP70) or enolase (ENO). Both proteins are missing from pf18 axonemes and greatly reduced in cpc1 axonemes. Gels were 7% acrylamide for A and C and the stained image in D and 10% acrylamide for the immunoblots in D. (E) Immunoblots of sucrose gradient fractions of cpc1 KI extracts were probed with the indicated antibodies and reveal the loss of enolase and HSP70A in fractions 5 and 6. The sizes (in kDa) of molecular mass standards are indicated next to each gel (A and D) and the enolase blot (D). The gel in A and the anti-CPC1 immunoblot in C appeared in a previous publication (Zhang and Mitchell, 2004). (F) Enolase activity in axonemal extracts. Enolase activity was measured in sucrose gradient fractions, similar to those shown in A, prepared with extracts from equal numbers of wild-type (•) or cpc1 (○) axonemes. Data are representative of two independent experiments.
Figure 3.
Figure 3.
Blot analysis of flagellar enolase in wild-type and central pair assembly defective mutants. (A) Stained gel of whole flagella (FLA), and the insoluble axoneme (AXO) and soluble membrane + matrix (M+M) fractions generated by treatment with NP-40 detergent. Samples were prepared from wild-type (W), cpc1 (C), or pf18 (P) cells. (B) Immunoblot of a gel identical to A except that sample loads were reduced 50-fold and the separating gel contained 1 M urea, probed with anti-enolase (C-19). The sizes (in kDa) of molecular mass standards are indicated next to the stained panel.
Figure 4.
Figure 4.
Diagram summarizing steps of glycolysis known to occur in the cytosol in Chlamydomonas, including the two steps that can contribute to ATP synthesis.
Figure 5.
Figure 5.
Identification of pyruvate kinase in flagellar fractions. (A) Sequence of the pyruvate kinase encoded by C_2590004, identified by proteomic analysis of flagellar extracts. Peptides identified by mass spectroscopy are underlined. Two of these (bold) were used to raise antibodies. (B) The C_2590004 gene product, PYK4, consists of four pyruvate kinase domains (PYK) separated by short spacers. (C) Gel stained with Coomassie blue (CB) and immunoblot (PYK4) of flagellar proteins (lanes F) showing the specificity of anti-PYK4 antibody 16562 for a 240-kDa band. (D) Gel (CB) and immunoblot (PYK4) of flagellar fractions probed with anti-PYK4 antibody 16562. Flagella (F) were fractionated into insoluble axonemal pellets (P) and soluble supernates (S) by either detergent or freeze-thaw treatments. The sizes (in kDa) of molecular mass standards (std) are indicated next to the stained panels.

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