Isolation and Proteomic Analysis of Chlamydomonas Centrioles
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 WordsCentrioles basal bodies Chlamydomonas proteomics MudPIT nephronophthisis Meckel Syndrome Oral-Facial-Digital Syndrome primary cilia PACRG
The authors thank Edwin Romijn and John R. Yates III for a highly productive collaboration and for many helpful discussions. This work was supported by NSF grant MCB0416310, NIH grant R01 GM077004-01A1, the Searle Scholars Program, a Hellman Family Award for Early Career Faculty, and a UCSF REAC award.
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