Comparative phosphoproteomics to identify targets of the clock-relevant casein kinase 1 in C. reinhardtii Flagella

Methods Mol Biol. 2014:1158:187-202. doi: 10.1007/978-1-4939-0700-7_12.

Abstract

In the green biflagellate alga Chlamydomonas reinhardtii different clock-relevant components have been identified that are involved in maintaining phase, period, and amplitude of circadian rhythms. It became evident that several of them are interconnected to flagellar function such as CASEIN KINASE1 (CK1). CK1 is involved in keeping the period. But it is also relevant for the formation of flagella, where it is physically located, and it controls the swimming velocity. In this chapter, we describe (1) how the flagellar sub-proteome is purified, (2) how phosphopeptides from this organelle are enriched, (3) how in vivo phosphorylation sites are determined, and (4) how direct and indirect flagellar targets of CK1 can be found using a specific inhibitor. Such a procedure can also be employed with other clock-relevant kinases if specific inhibitors or mutants are available.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Casein Kinase I / metabolism*
  • Chlamydomonas reinhardtii / genetics
  • Chlamydomonas reinhardtii / metabolism*
  • Circadian Clocks / physiology*
  • Flagella*
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Proteome
  • Proteomics* / methods

Substances

  • Phosphoproteins
  • Proteome
  • Casein Kinase I