|Home | About | Journals | Submit | Contact Us | Français|
Chlamydomonas flagella contain a molecular chaperone now identified as HSP70A, a major cytoplasmic isoform. HSP70A synthesis is upregulated by deflagellation, and its distribution in the flagellum overlaps with the IFT kinesin-II motor FLA10. HSP70A may chaperone flagellar proteins during transport, participating in the assembly and maintenance of the flagellum.
The multiple compartments of the eukaryotic cell contain Hsp70 molecular chaperones that participate in the folding, targeting, assembly, and maintenance of the proteome (2, 9, 27). We previously found a putative flagellar Hsp70 in the green alga Chlamydomonas reinhardtii (1). Eukaryotic flagella are highly specialized, motile structures thought to contain only proteins directly involved in their assembly and function (6, 10, 17, 21). We identified this flagellar protein as Chlamydomonas HSP70A, a cytoplasmic chaperone, and examined its expression and localization within the context of the flagellum.
The flagellar protein recognized by the pan-Hsp70 monoclonal antibody MA3-006 (Affinity BioReagents, Denver, CO) (1) was isolated from the matrix fraction of Chlamydomonas flagella (strain CC-1690; Chlamydomonas Genetics Center, Durham, NC) (26) by binding to ATP-agarose (Sigma, St. Louis, MO) (25), followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. (Fig.1).1). Following tryptic digestion on a blot, matrix-assisted laser desorption-time of flight mass spectrometry (Keck Protein Chemistry Laboratory, University of Massachusetts Medical School, Worcester, MA) uniquely identified the protein as HSP70A (13, 24), matching 9 of 11 peptide masses covering 26.2% of the protein. HSP70A is one of seven Chlamydomonas Hsp70 family members (20) and bears the carboxy-terminal EEVD invariably conserved in cytoplasmic isoforms (8).
Genes encoding flagellar proteins typically are transcriptionally upregulated during organellar assembly (6, 10, 17, 21). Total RNAsamples were isolated (QIAGEN, Valencia, CA) from cells (CC-3941) bearing full-length flagella and cells with half-length, actively regenerating flagella (30 min after pH shock deflagellation). Reverse transcription (RT)-PCR (Titan; Roche, Indianapolis, IN) was performed using mRNA-specific primers (MWG, High Point, NC; for alpha-tubulin, GCCGGTATCCAGGTGGGCAATG and GATCAGCTGCTCGGGGTGGAAC; for beta-tubulin, CTGGAGCGCATCAACGTGTACTTC and CCTGGAAGCCCTGCAGGCAG; for HSP70A, CATACGCGACTATTCTGCCGCTATAC and GGGTTCATAGCGACCTGGTTCTTG; for CRY1, CCCAAGGAGGTGGTGAGCCTG and GATGCCCAGCTCCTTGCACTTC; for CBLP, GGCCAGTTCTGCCTGACTG and CGACCACGATCTGGCGGTTGTC; and for HSP70B, GTGCTGAGAAGGTCGTGGGTATC and CCTCAATCACCCGGTAAGGCAC) in five-cycle increments. Products were sized on agarose gels and documented using an AlphaImager (AlphaInnotech, San Leandro, CA). HSP70A mRNA levels increased strongly during flagellar assembly along with tubulin mRNAs (Fig. (Fig.2)2) (22), while constitutively expressed messages for ribosomal protein S14 (14) and G protein beta subunit-like protein (18) remained unchanged. However, HSP70B mRNAs, encoding a chloroplast-localized isoform (5), were also elevated, suggesting that HSP70A upregulation may be part of a whole-cell stress response to deflagellation (22).
To study the cellular distribution of HSP70A, affinity-purified antipeptide antibodies were generated (Research Genetics, Huntsville, AL) to its unique carboxy terminus (PSGGSGAGPKIEEVD) (13, 24). These antibodies reacted specifically with a 70-kDa protein on immunoblots of total cell protein (TP) and purified flagellar protein (FL) that was also recognized by MA3-006 (1) (Fig. (Fig.3).3). Based upon a yield of 10 μg of flagellar protein per 107 cells (26), flagella contain approximately 10% of the cellular HSP70A pool. In contrast, antibodies to chloroplast-localized HSP70B show a strong reaction to a cell body protein not present in flagella. Recent global characterization of the flagellar proteome by mass spectrometry also identified HSP70A as a Chlamydomonas flagellar protein (G. Pazour, N. Agrin, and G. B. Witman, unpublished data; cited at http://genome.jgi-psf.org/cgi-bin/dispGeneModel?db=chlre2&tid=155023).
In addition, an epitope-tagged HSP70A allele was constructed. Complementary short oligonucleotides (TCGACTACCCCTACGACGTCCCCGACTACGCCGGCGAGGAGGTGGACTAA and TCGATTAGTCCACCTCCTCGCCGGCGTAGTCGGGGACGTCGTAGGGGTAG) encoding a hemagglutinin (HA) epitope (7) and retaining the highly conserved terminal sequence EEVD (8) were synthesized, annealed, and cloned into a SalI site in the HSP70A gene (13, 24) to append the carboxy-terminal sequence YPYDVPDYAGEEVD. This construct was cotransformed into Chlamydomonas cells (CC-2929) (14) with a selectable marker. HSP70A::HA was detected in roughly 10% of transformants by using the anti-HA monoclonal antibody 12CA5 (Roche). Several clones with stable, high levels of expression were characterized. Ectopic expression of HSP70::HA did not have gross effects on either growth or motility. On immunoblots using MA3-006 (which binds an internal sequence unchanged by epitope addition), HSP70A and HSP70A::HA were present at similar levels in transformant total cell protein and flagellar protein samples (Fig. (Fig.3).3). Note that HSP70A and HSP70A::HA can be distinguished immunologically using antipeptide and antiepitope antibodies, respectively, because of the carboxy-terminal placement of the epitope.
Immunofluorescent localization (11) of HSP70A confirmed that the chaperone was abundant in cell body cytoplasm and present in flagella (Fig. (Fig.4).4). Within flagella, wild-type HSP70A was distributed in a discontinuous, punctate fashion and concentrated in flagellar tips (Fig. (Fig.4).4). Previous localizations with MA3-006 (1) showed primarily tip labeling; the difference may be due to improvements in imaging technology (push-processed film versus a cooled charge-coupled-device camera) and antibody affinity. HSP70A::HA was distributed along the flagellum like the wild-type protein, although accumulation in the flagellar tip was less evident. Interestingly, in HSP70A::HA, the epitope coincides with a cochaperone binding site (8), implicating regulatory interaction and substrate release in its accumulation in the tip.
The flagellar distribution of HSP70A is very similar to that of the components of the intraflagellar transport (IFT) system (3, 17, 19) responsible for shuttling unassembled axonemal proteins from the cell body to the flagellar tips. Immunofluorescent colocalization (11) of HSP70A (using the rabbit antipeptide antibodies; red channel) and the anterograde IFT kinesin-II FLA10 (using mouse monoclonal antibody K2.4 [Berkeley Antibody Co, Richmond, CA]; green channel) (4, 12, 16) showed similar overlapping but nonidentical distributions (Fig. 5B and C). The overwhelming majority (>90%) of FLA10 concentrations were associated with HSP70A. HSP70A concentrations were rarely observed without an associated FLA10 signal (shown most clearly in the blue colocalization analysis), although the stoichiometry of labeling (indicated by yellow hues in red-green overlays) varied considerably (Fig. 5D and E). This variation suggests that HSP70A is not an integral part of the IFT machinery but is instead carried by IFT as cargo. Despite considerable effort, we have been unable to identify specific HSP70A interactors by cofractionation, native gel electrophoresis, immunoprecipitation, cross-linking, or immunoelectron microscopy approaches, although flagellar Hsp70 was reported to copurify with IFT complexes on sucrose gradients in one study (15; see also reference 4). Within the flagellum, HSP70A may be interacting with many different molecular targets (redistributing as conditions change), which is consistent with the generalist strategy of Hsp70 involvement in protein folding, transport, and repair observed in other systems (2, 9, 27). Hsp70 proteins have also been implicated directly in cargo release from kinesin-driven anterograde fast axonal transport (23), suggesting that HSP70A may assist with both folding and delivery of flagellar proteins. Our molecular identification of this molecular chaperone opens the door to further investigation of its roles in flagellar assembly/disassembly and maintenance.
We thank Christoph Beck, Paul Lefebvre, and Elizabeth Harris for sharing Chlamydomonas strains and plasmids, John Leszyk for assistance with the matrix-assisted laser desorption-time of flight analysis, Marina del Rios for help with Chlamydomonas transformation, Geraldine Sheir-Neiss for technical assistance, John Butler for laboratory support, several reviewers for their constructive comments, and Wayne Rasband and Christopher Philip Mauer for making available the ImageJ program and RG2B colocalization plug-in, respectively.
This work was supported by a fellowship (to J.S.) from the Howard Hughes Institute for Undergraduate Biological Sciences Medical Research Program (to Haverford College), the Haverford College Provost's Office, and NSF MCB-9506236 and MCB-9982733 (to K.A.J.).