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. 2007 Jun;18(6):2336-45.
doi: 10.1091/mbc.e07-01-0069. Epub 2007 Apr 11.

Functional interaction between phosducin-like protein 2 and cytosolic chaperonin is essential for cytoskeletal protein function and cell cycle progression

Peter C Stirling  1 , Martin Srayko, Karam S Takhar, Andrei Pozniakovsky, Anthony A Hyman, Michel R Leroux
Affiliations

Functional interaction between phosducin-like protein 2 and cytosolic chaperonin is essential for cytoskeletal protein function and cell cycle progression

Peter C Stirling et al. Mol Biol Cell. 2007 Jun.

Abstract

The Chaperonin Containing Tcp1 (CCT) maintains cellular protein folding homeostasis in the eukaryotic cytosol by assisting the biogenesis of many proteins, including actins, tubulins, and regulators of the cell cycle. Here, we demonstrate that the essential and conserved eukaryotic phosducin-like protein 2 (PhLP2/PLP2) physically interacts with CCT and modulates its folding activity. Consistent with this functional interaction, temperature-sensitive alleles of Saccharomyces cerevisiae PLP2 exhibit cytoskeletal and cell cycle defects. We uncovered several high-copy suppressors of the plp2 alleles, all of which are associated with G1/S cell cycle progression but which do not appreciably affect cytoskeletal protein function or fully rescue the growth defects. Our data support a model in which Plp2p modulates the biogenesis of several CCT substrates relating to cell cycle and cytoskeletal function, which together contribute to the essential function of PLP2.

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Figures

Figure 1.
Figure 1.
PhLP2/PLP2 is an essential CCT-binding protein. (A) CCT2-HA is immunoprecipitated using an anti-myc antibody from S. cerevisiae cells coexpressing CCT-HA and plp2-myc but not from control wild-type (WT) or CCT-HA–expressing cells. HA-tagged CCT is detected by Western blot analysis by using an anti-HA antibody. (B) Western blot showing CCT coprecipitating in vitro with GST-PhLP2A or GST-PhLP3 in a GST pull-down, but not the GST-alone control. Purified CCT was run as a control to show that the antibody recognizes multiple CCT subunits (left). (C) Counterselection of a URA3-marked plasmid carrying PLP2 ([pURA3 PLP2]) in plp2Δ cells with 5′-fluoroorotic acid (5′FOA) shows that PLP2 is an essential gene. Cells prototrophic (PLP2 URA3) and auxotrophic (PLP2 ura3-52) for uracil (ura) were included as controls. SC, synthetic complete media.
Figure 2.
Figure 2.
Generation and analysis of temperature-sensitive plp2 alleles. (A) Wild-type yeast (PLP2), or two temperature-sensitive alleles of PLP2, namely, plp2-1 and plp2-2, were spotted on YPD media and grown at the temperatures shown for 48 h. plp2-1 displays a more severe ts phenotype than plp2-2. (B) Reversibility of the plp2-ts alleles was assessed after growth at 37°C for the times shown. Shifted cells were plated at a permissive temperature and the number of microcolony forming cells was assessed under a microdissection microscope. plp2-2 temperature sensitivity is largely reversible compared with the more severe plp2-1 allele.
Figure 3.
Figure 3.
plp2-ts cells have increased sensitivity to cytoskeletal-destabilizing drugs and are larger than wild-type cells. (A) Benomyl and latrunculin sensitivity of plp2-1 and plp2-2 mutants relative to wild-type (PLP2) cells, as determined by relative clearance caused by drug-inoculated paper discs when grown at the semipermissive temperature of 30°C. (B) Cell sizes of the indicated strains were measured perpendicular to the mother-daughter axis for at least 100 cells. An asterisk (*) indicates that, relative to wild type, the cells were significantly larger (p < 0.01) as determined by an independent variable t test.
Figure 4.
Figure 4.
plp2-ts cells exhibit aberrant nuclear segregation and spindle orientation. (A) Images of DAPI-stained PLP2, plp2-1 and plp2-2 cells grown at permissive (23°C) and nonpermissive (37°C) temperatures. Arrows indicate multinucleate cells. (B) GFP-α-tubulin–expressing PLP2, plp2-1, and plp2-2 cells were visualized and scored for spindle orientation with respect to the mother-daughter axis. Cells are outlined in white and the percentages of misoriented spindles are indicated below each panel. Representative images are shown of normal spindles for wild-type and plp2-1 and plp2-2 cells at 23°C and of misoriented spindles in plp2-1 and plp2-2 at 37°C.
Figure 5.
Figure 5.
Actin filament organization defects in plp2-ts cells. (A) PLP2, plp2-1, and plp2-2 cells grown at permissive temperature (25°C) or nonpermissive temperature (37°C) were stained with anti-actin antibodies. Arrows indicate examples of cells lacking clear actin cables and having poorly polarized actin patches.
Figure 6.
Figure 6.
Mammalian PhLP2A modulates CCT-mediated actin folding in vitro. (A) In vitro folding reactions of nascent 35S-labeled actin in the presence of GST or GST-PhLP2A and separated on a nondenaturing gel at the time points (minutes) indicated. CCT:actin and PFD:actin binary complexes, as well as native actin, are indicated. The identity of the fast-migrating band as native monomeric actin was verified by Dnase I-shifting on the native gel (right). Asterisk (*) indicates Dnase I bound native actin. Note the reduction in the intensity of the bottom band. Note the decrease in native actin production over time in the presence of GST-PhLP2A but not GST alone (see Supplemental Figure S3 for quantitation of band intensities). An SDS-gel illustrating the relative amounts of translation products is shown (bottom). (B) Coprecipitation of 35S-labeled actin or β-tubulin with GST-PhLP2A in a reticulocyte translation reaction. The lower panel shows that the levels of translation were comparable. (C) Coprecipitation of 35S-labeled actin produced in E. coli lysate takes place only in the presence of GST-PhLP2A and exogenously added CCT, indicating the formation of a ternary complex. Actin cDNA was translated in E. coli lysate with or without the addition of purified rabbit CCT, and the relative levels of the translation products are shown on the left. GST-PhLP2A coprecipitated the newly-made actin only in reactions to which CCT had been added, whereas GST alone had no such affect as shown on the right.
Figure 7.
Figure 7.
High-copy suppressors of plp2-1 indicate a role for PLP2 in cell cycle progression. Partial suppressors of plp2-1 temperature sensitivity identified in a high-copy screen (PLC1 suppression is shown in Supplemental Figure S4) (A) and suppressors identified in the literature (B). plp2-1 cells carrying the indicated plasmids were grown to log phase and serially diluted on SC-Ura media and grown for 2–3 d at permissive (25°C) or restrictive (37°C) temperatures before imaging. (C) A delay in DNA replication is observed in synchronized plp2-1 cells. Graphs indicate FACS analysis of cells stained for DNA content over time. (D) Allele specific suppression of plp2-ts alleles. Serially diluted plp2-2 cultures carrying the indicated plasmids were grown for 2–3 d on SC-Ura as in A and B before imaging.
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References

    1. Adams A., Gottschling D. E., Kaiser C. A., Stearns T. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1997. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual.
    1. Amberg D. C., Burke D. J., Strathern J. N. 2005 ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2005. Methods in Yeast Genetics.
    1. Ayscough K. R., Stryker J., Pokala N., Sanders M., Crews P., Drubin D. G. High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A. J. Cell Biol. 1997;137:399–416. - PMC - PubMed
    1. Blaauw M., Knol J. C., Kortholt A., Roelofs J., Ruchira , Postma M., Visser A. J., van Haastert P. J. Phosducin-like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins. EMBO J. 2003;22:5047–5057. - PMC - PubMed
    1. Camasses A., Bogdanova A., Shevchenko A., Zachariae W. The CCT chaperonin promotes activation of the anaphase-promoting complex through the generation of functional Cdc20. Mol. Cell. 2003;12:87–100. - PubMed

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