Isolation and Proteomic Analysis of Chlamydomonas Centrioles

  • Lani C Keller
  • Wallace F Marshall
Part of the Methods in Molecular Biology™ book series (MIMB, volume 432)

Summary

Centrioles are barrel-shaped cytoskeletal organelles composed of nine triplet microtubules blades arranged in a pinwheel-shaped array. Centrioles are required for recruitment of pericentriolar material (PCM) during centrosome formation, and they act as basal bodies, which are necessary for the outgrowth of cilia and flagella. Despite being described over a hundred years ago, centrioles are still among the most enigmatic organelles in all of cell biology. To gain molecular insights into the function and assembly of centrioles, we sought to determine the composition of the centriole proteome. Here, we describe a method that allows for the isolation of virtually “naked” centrioles, with little to no obscuring PCM, from the green alga, Chlamydomonas. Proteomic analysis of this material provided evidence that multiple human disease gene products encode protein components of the centriole, including genes involved in Meckel syndrome and Oral-Facial-Digital syndrome. Isolated centrioles can be used in combination with a wide variety of biochemical assays in addition to being utilized as a source for proteomic analysis.

Key Words

Centrioles basal bodies Chlamydomonas proteomics MudPIT nephronophthisis Meckel Syndrome Oral-Facial-Digital Syndrome primary cilia PACRG 

References

  1. 1.
    Marshall, W. F. (2001) Centrioles take center stage. Curr. Biol. 11, R487–R496.CrossRefPubMedGoogle Scholar
  2. 2.
    Keller, L. C., Romijn, E. P., Zamora, I. Yates, J. R., and Marshall, W. F. (2005) Proteomic analysis of isolated Chlamydomonas centrioles reveals orthologs of ciliary disease genes. Curr. Biol. 15, 1090–1098.CrossRefPubMedGoogle Scholar
  3. 3.
    Doxsey, S., Zimmerman, W., and Mikule, K. (2005). Centrosome control of the cell cycle. Trends Cell Biol. 15, 303–311.CrossRefPubMedGoogle Scholar
  4. 4.
    Li, J. B., Gerdes, J. M, Haycraft, C. J., Fan, Y., Teslovich, T. M., May-Simera, H., et al. (2004). Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene. Cell 117, 541–552.CrossRefPubMedGoogle Scholar
  5. 5.
    Andersen, J. S., Wilkinson, C. J., Mayor, T., Mortensen, P., Nigg, E., and Mann, M. (2003). Proteomic characterization of the human centrosome by protein correlation profiling. Nature 426, 570–574.CrossRefPubMedGoogle Scholar
  6. 6.
    Snell, W. J, Dentler, W. L., Haimo, L. T., Binder, L. I., Rosenbaum, J. L. (1974). Assembly of chick brain tubulin onto isolated basal bodies of Chlamydomonas reinhardtii. Science 185, 357–360.CrossRefPubMedGoogle Scholar
  7. 7.
    Washburn, M. P., Wolters, D., and Yates, J. R. III (2001). Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247.CrossRefPubMedGoogle Scholar
  8. 8.
    Harris, E. H. (1989). Procedures and Resources The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press Inc., San, Diego, CA, USA pp. 578–579.Google Scholar
  9. 9.
    Eng, J., McCormack, A., and Yates, J. R. (1994). An approach to correlate tandem mass-spectral data of peptides with amino-acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989.CrossRefGoogle Scholar
  10. 10.
    Stolc, V., Samanta, M. P., Tongprasit, W., and Marshall, W. F. (2005). Genome-wide trancriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes. Proc. Natl. Acad. Sci. U.S.A. 102, 3703–3707.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Lani C Keller
    • 1
  • Wallace F Marshall
    • 1
  1. 1.Department of Biochemistry & BiophysicsUniversity of CaliforniaSan FranciscoCA

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