Analysis of interactions between intraflagellar transport proteins.

Intraflagellar transport (IFT) involves the movement of large proteinaceous particles or trains along the length of ciliary and flagellar axonemal microtubules. The particles contain multiple copies of two protein complexes. As isolated from the flagellated model organism, Chlamydomonas reinhardtii, IFT A contains 6 distinct gene products while IFT B contains at least 13 distinct gene products. To better understand the architecture of these two complexes, a multifaceted approach has been employed to identify subcomplexes and specific protein-protein interactions. The high biochemical yields afforded with Chlamydomonas preparations have allowed traditional biochemical approaches including chemical cross-linking and disruption of native complexes, which, in the case of IFT B, have revealed a core subcomplex retaining nine of the B subunits. Complementing these results are molecular approaches including two-hybrid screenings and heterologous expression that have identified specific protein-protein interactions. Lastly, genetic approaches utilizing Chlamydomonas IFT mutants have shown how the loss of specific subunits perturb the complexes and, in the case of IFT A, they have revealed a core subcomplex containing half of the A subunits.

Subunit interactions and organization of the Chlamydomonas reinhardtii intraflagellar transport complex A proteins.

Chlamydomonas reinhardtii intraflagellar transport (IFT) particles can be biochemically resolved into two smaller assemblies, complexes A and B, that contain up to six and 15 protein subunits, respectively. We provide here the proteomic and immunological analyses that verify the identity of all six Chlamydomonas A proteins. Using sucrose density gradient centrifugation and antibody pulldowns, we show that all six A subunits are associated in a 16 S complex in both the cell bodies and flagella. A significant fraction of the cell body IFT43, however, exhibits a much slower sedimentation of ∼2 S and is not associated with the IFT A complex. To identify interactions between the six A proteins, we combined exhaustive yeast-based two-hybrid analysis, heterologous recombinant protein expression in Escherichia coli, and analysis of the newly identified complex A mutants, ift121 and ift122. We show that IFT121 and IFT43 interact directly and provide evidence for additional interactions between IFT121 and IFT139, IFT121 and IFT122, IFT140 and IFT122, and IFT140 and IFT144. The mutant analysis further allows us to propose that a subset of complex A proteins, IFT144/140/122, can form a stable 12 S subcomplex that we refer to as the IFT A core. Based on these results, we propose a model for the spatial arrangement of the six IFT A components.

The RABL5 homolog IFT22 regulates the cellular pool size and the amount of IFT particles partitioned to the flagellar compartment in Chlamydomonas reinhardtii.

Cilia and flagella, sensory and motile structures protruding from the cell body, rely on the continuous bidirectional traffic of intraflagellar transport (IFT) particles to ferry flagellar precursors into flagella for assembly. Cells synthesize a large pool of IFT particle proteins in the cell body, but only a small portion engages in active transport within the flagella at any given time. The atypical small G protein Rab-like 5 (RABL5) has been shown to move in an IFT-like manner in the flagella, but its function in ciliogenesis is controversial. In this report, we demonstrate that IFT22, the Chlamydomonas reinhardtii homolog of RABL5, is a bona fide IFT particle complex B subunit. Although the amount of IFT22 remains unaffected by depletion of either complex A or B, depletion of IFT22 leads to a smaller pool of both complex A and B. Strikingly, the smaller cellular pool of IFT particles does not lead to a reduced distribution of IFT particles to flagella. Instead, the amount of IFT particle proteins, including IFT22 itself, increase in the flagella. Moreover, cells over-expressing IFT22 also accumulate IFT particles in their flagella. Taken together, these data indicate that, in C. reinhardtii, IFT22 controls the cellular levels of both complex A and B, thus plays a critical role in determining the cellular availability of IFT particles. In addition, although IFT22 may not directly carry any precursors for flagellar assembly, it controls how many IFT particles participate in ferrying precursors into flagella.

Chlamydomonas IFT70/CrDYF-1 is a core component of IFT particle complex B and is required for flagellar assembly.

DYF-1 is a highly conserved protein essential for ciliogenesis in several model organisms. In Caenorhabditis elegans, DYF-1 serves as an essential activator for an anterograde motor OSM-3 of intraflagellar transport (IFT), the ciliogenesis-required motility that mediates the transport of flagellar precursors and removal of turnover products. In zebrafish and Tetrahymena DYF-1 influences the cilia tubulin posttranslational modification and may have more ubiquitous function in ciliogenesis than OSM-3. Here we address how DYF-1 biochemically interacts with the IFT machinery by using the model organism Chlamydomonas reinhardtii, in which the anterograde IFT does not depend on OSM-3. Our results show that this protein is a stoichiometric component of the IFT particle complex B and interacts directly with complex B subunit IFT46. In concurrence with the established IFT protein nomenclature, DYF-1 is also named IFT70 after the apparent size of the protein. IFT70/CrDYF-1 is essential for the function of IFT in building the flagellum because the flagella of IFT70/CrDYF-1-depleted cells were greatly shortened. Together, these results demonstrate that IFT70/CrDYF-1 is a canonical subunit of IFT particle complex B and strongly support the hypothesis that the IFT machinery has species- and tissue-specific variations with functional ramifications.

Purification of IFT particle proteins and preparation of recombinant proteins for structural and functional analysis.

Intraflagellar transport (IFT) is characterized by a robust bidirectional movement of large proteinaceous particles along the length of eukaryotic cilia and flagella. Essential for the assembly and function of the organelle, IFT is believed to transport a large array of ciliary components in and out of the organelle. Biochemical analysis of the proteins involved with this transport has been largely dependent on the ability to isolate suitable quantities of intact cilia or flagella. One model organism, Chlamydomonas reinhardtii, has proven to be especially well-suited for such endeavors. Indeed, many of the IFT particle proteins were initially identified through biochemical analysis of green algae. This chapter describes some of the most effective methods for the purification of IFT particle proteins from Chlamydomonas flagella. This chapter also describes complementary approaches where recombinant IFT proteins are generated with affinity tags that allow rapid and specific purification. The recombinant proteins can be used to analyze protein-protein interactions and can be directly delivered to mutant cells to analyze functional domains. Although the techniques described here are focused entirely on Chlamydomonas IFT proteins, the approaches, especially regarding recombinant proteins, should be applicable to the study of IFT machinery in other model organisms.

Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions.

The molecular chaperone Hsp (heat-shock protein) 90 is critical for the activity of diverse cellular client proteins. In a current model, client proteins are transferred from Hsp70 to Hsp90 in a process mediated by the co-chaperone Sti1/Hop, which may simultaneously interact with Hsp70 and Hsp90 via separate TPR (tetratricopeptide repeat) domains, but the mechanism and in vivo importance of this function is unclear. In the present study, we used truncated forms of Sti1 to determine the minimal regions required for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. We found that both TPR1 and TPR2B contribute to the Hsp70 interaction in vivo and that mutations in both TPR1 and TPR2B were required to disrupt the in vitro interaction of Sti1 with the C-terminus of the Hsp70 Ssa1. The TPR2A domain was required for the Hsp90 interaction in vivo, but the isolated TPR2A domain was not sufficient for the Hsp90 interaction unless combined with the TPR2B domain. However, isolated TPR2A was both necessary and sufficient for purified Sti1 to migrate as a dimer in solution. The DP2 domain, which is essential for in vivo function, was dispensable for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. As evidence for the role of Sti1 in mediating the interaction between Hsp70 and Hsp90 in vivo, we identified Sti1 mutants that result in reduced recovery of Hsp70 in Hsp90 complexes. We also identified two Hsp90 mutants that exhibit a reduced Hsp70 interaction, which may help clarify the mechanism of client transfer between the two molecular chaperones.

Characterization of the intraflagellar transport complex B core: direct interaction of the IFT81 and IFT74/72 subunits.

Required for the assembly and maintenance of eukaryotic cilia and flagella, intraflagellar transport (IFT) consists of the bidirectional movement of large protein particles between the base and the distal tip of the organelle. Anterograde movement of particles away from the cell body is mediated by kinesin-2, whereas retrograde movement away from the flagellar tip is powered by cytoplasmic dynein 1b/2. IFT particles contain multiple copies of two distinct protein complexes, A and B, which contain at least 6 and 11 protein subunits, respectively. In this study, we have used increased ionic strength to remove four peripheral subunits from the IFT complex B of Chlamydomonas reinhardtii, revealing a 500-kDa core that contains IFT88, IFT81, IFT74/72, IFT52, IFT46, and IFT27. This result demonstrates that the complex B subunits, IFT172, IFT80, IFT57, and IFT20 are not required for the core subunits to stay associated. Chemical cross-linking of the complex B core resulted in multiple IFT81-74/72 products. Yeast-based two-hybrid and three-hybrid analyses were then used to show that IFT81 and IFT74/72 directly interact to form a higher order oligomer consistent with a tetrameric complex. Similar analysis of the vertebrate IFT81 and IFT74/72 homologues revealed that this interaction has been evolutionarily conserved. We hypothesize that these proteins form a tetrameric complex, (IFT81)2(IFT74/72)2, which serves as a scaffold for the formation of the intact IFT complex B.

Role of acetyl-coenzyme A synthetase in leaves of Arabidopsis thaliana.

Acetyl-coenzyme A synthetase (ACS) is a plastidic enzyme that forms acetyl-coenzyme A (acetyl-CoA) from acetate and coenzyme A using the energy from ATP. Traditionally it has been thought to be the major source for the production of acetyl-CoA destined for fatty acid formation. Recent work suggested that the accumulation of lipids in developing Arabidopsis seeds was more closely correlated with the expression of pyruvate dehydrogenase complex than with the expression of ACS, suggesting that most of the carbon for fatty acid formation in the plastids of seeds comes from pyruvate rather than from acetate. To explore the role of this enzyme, Arabidopsis plants with altered amounts of ACS were generated by overexpressing its cDNA in either the sense or the antisense configuration. The resulting plants had in vitro enzyme activities that ranged from about 5% to over 400% of wild-type levels. The rate of [1-14C]acetate conversion into fatty acids was closely related to the in vitro ACS activity, showing that the amount of enzyme clearly limited the capacity of leaves to convert exogenous acetate to fatty acids. There was, however, no relationship between the ACS level and the capacity of the plants to incorporate 14CO2 into 14C-labeled fatty acids. These data strongly support the idea that, although plants can convert acetate into fatty acids, relatively little carbon moves through this pathway under normal conditions.