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Analysis of Eukaryotic Molecular Chaperone Complexes Involved in Actin Folding

  • Michel R. Leroux
Part of the Methods in Molecular Biology™ book series (MIMB, volume 140)

Abstract

Actin, the most abundant protein of eukaryotes, plays critical roles in muscle contraction and in the movement of and within cells, including ameboid locomotion, protoplasmic streaming, migration of cellular organelles and chromosomes, formation of cell evaginations, and stabilization of cell shape. The dynamics of actin filament assembly and disassembly, as well as the organization of the actin cytoskeleton into bundles and networks of filaments, are highly complex, being regulated by a large number of actin crosslinking, capping, and severing proteins (1, 2). Interestingly, the biogenesis of actin also depends on at least two accessory proteins; the first to be described is the eukaryotic cytosolic chaperonin known as TCP-1 ring complex (TRiC) or chaperonin-containing TCP-1 (CTT), which is also involved in tubulin biogenesis (3, 4, 5, 6, 7, 8, 9, 10). A second component involved in actin biogenesis has been recently identified independently by two groups. In a screen for S. cerevisiae mutants that are synthetically lethal with a mutant allele of γ-tubulin, Geissler et al. (11) identified five genes that encode proteins that assemble into a heterooligomeric complex named Gim-complex (GimC; Gim is an acronym for genes involved in microtubule biogenesis). Deletion of GIM genes causes both microtubule and actin cytoskeletal defects, a property also observed for genes encoding defective TRiC subunits (9 12, 13, 14). In addition, synthetic lethality is observed with certain combinations of GIM and TRiC gene mutations. Vainberg et al. (15) reported on the purification of the mammalian homolog of GimC from bovine testis based on its ability to form a stable binary complex with unfolded ß-actin. The chaperone complex, named prefoldin (PFD) because of its ability to transfer unfolded actin to the chaperonin TRiC for subsequent folding to the native state, consists of six proteins, PFD1-6. Although TRiC is apparently both necessary and sufficient for the ATP-dependent folding of actin in vitro, it is likely that in vivo both GimC/PFD and TRiC cooperate to ensure that actin reaches its native state following its synthesis. Additional components may be involved, for instance, an unidentified protein that for the purpose of this chapter, is referred to as IBP (intermediate mobility actin binding protein) because of its ability to form a complex with denatured actin that migrates on a native gel between the TRiC-actin and GimC-actin complexes. Here, methods for the detection and analysis of unfolded actin binding proteins are presented. In Chapters 17, 20, and 21, protocols for purifying TRiC from bovine testis, as well as GimC/PFD from S. cerevisiae and bovine testis are detailed.

Keywords

Actin Binding Rabbit Reticulocyte Lysate Protoplasmic Streaming Bovine Testis Actin Filament Assembly 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Michel R. Leroux
    • 1
  1. 1.Department of Cellular BiochemistryMax Planck Institute of BiochemistryMartinsriedGermany

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