Mutational analysis of capping protein function in Saccharomyces cerevisiae.

Sizonenko, Gennady I.; Karpova, Tatiana S.; Gattermeir, David J.; Cooper, John A.

doi:10.1091/mbc.7.1.1

Cited by 29 publications

(31 citation statements)

References 32 publications

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“…The crystal structure of chicken CapZ (␣1␤1) demonstrates that the C termini are amphipathic helices that probably form contacts with two separate actin monomers (21). This interpretation is consistent with earlier experimental evidence demonstrating the importance of the C terminus from the ␤-subunit (44,45,48,53).…”

Section: Discussionsupporting

confidence: 86%

“…No obvious candidates for a rice ␤-subunit of capping protein were found, however. Mutagenesis studies and the recent crystal structure of chicken muscle CapZ ␣1/␤1 heterodimer implicate the C-terminal regions of both subunits in binding to F-actin (21,44,45). It is noteworthy that these regions are quite poorly conserved in AtCPA and AtCPB.…”

Section: Identification Of Plant Cappingmentioning

confidence: 99%

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Arabidopsis Capping Protein (AtCP) Is a Heterodimer That Regulates Assembly at the Barbed Ends of Actin Filaments

Huang

Blanchoin

Kovar

et al. 2003

Journal of Biological Chemistry

137

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The precise regulation of actin filament polymerization and depolymerization is essential for many cellular processes and is choreographed by a multitude of actinbinding proteins (ABPs). In higher plants the number of well characterized ABPs is quite limited, and some evidence points to significant differences in the biochemical properties of apparently conserved proteins. Here we provide the first evidence for the existence and biochemical properties of a heterodimeric capping protein from Arabidopsis thaliana (AtCP). The purified recombinant protein binds to actin filament barbed ends with K d values of 12-24 nM, as assayed both kinetically and at steady state. AtCP prevents the addition of profilin actin to barbed ends during a seeded elongation reaction and suppresses dilution-mediated depolymerization. It does not, however, sever actin filaments and does not have a preference for the source of actin. During assembly from Mg-ATP-actin monomers, AtCP eliminates the initial lag period for actin polymerization and increases the maximum rate of polymerization. Indeed, the efficiency of actin nucleation of 0.042 pointed ends created per AtCP polypeptide compares favorably with mouse CapZ, which has a maximal nucleation of 0.17 pointed ends per CapZ polypeptide. AtCP activity is not affected by calcium but is sensitive to phosphatidylinositol 4,5-bisphosphate. We propose that AtCP is a major regulator of actin dynamics in plant cells that, together with abundant profilin, is responsible for maintaining a large pool of actin subunits and a surprisingly small population of F-actin.The cytoskeleton in plant cells comprises a dynamic network of actin filaments, microtubules, and accessory proteins that powers cytoplasmic streaming, responds rapidly to prevent fungal attack, patterns the deposition of cellulosic wall polymers, and shapes cellular morphogenesis by choreographing exo-and endocytotic vesicle traffic. Understanding how the cytoskeleton is organized, how it responds to extrinsic and intrinsic cues, and how dynamics are regulated are central questions in plant biology (1). To date less than a dozen of the more than 70 classes of actin-binding proteins (ABPs) 1 found in eukaryotic cells (2, 3) have been isolated and characterized from plants. Particularly well studied are members of the profilin, ADF/cofilin, fimbrin, villin, EF-1␣, and myosin families (4, 5). Although the general properties of ABPs are often conserved across kingdoms, important differences highlight the need to examine in detail the functional properties of proteins from divergent organisms. For example, Chlamydomonas profilin binds extremely poorly to one of the major known ligands (poly-L-proline) and inhibits nucleotide exchange on actin (6), and phylogenetically unique classes of plant myosins are the fastest molecular motors on the planet (7). Based on genome sequencing, many classes of ABP are missing from higher plants, including ␣-actinin, spectrin, tropomyosin, WASp, and coronin (4,8). One intriguing possibility is that multiple iso...

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Section: Discussionsupporting

confidence: 86%

Section: Identification Of Plant Cappingmentioning

confidence: 99%

Arabidopsis Capping Protein (AtCP) Is a Heterodimer That Regulates Assembly at the Barbed Ends of Actin Filaments

Huang

Blanchoin

Kovar

et al. 2003

Journal of Biological Chemistry

137

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“…In other species, CP is an obligate heterodimer, and neither subunit alone is sufficient to bind actin (Amatruda et al, 1992;Sizonenko et al, 1996;Wear et al, 2003;Kim et al, 2004). To disrupt both subunits and further reduce functional CP protein levels, two homozygous double mutants (cpa-1 cpb-1 and cpa-1 cpb-3) were generated.…”

Section: Arabidopsis Cp Mutants Show Abnormal Cell and Organ Growthmentioning

confidence: 99%

Capping Protein Modulates the Dynamic Behavior of Actin Filaments in Response to Phosphatidic Acid inArabidopsis

et al. 2012

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Remodeling of actin filament arrays in response to biotic and abiotic stimuli is thought to require precise control over the generation and availability of filament ends. Heterodimeric capping protein (CP) is an abundant filament capper, and its activity is inhibited by membrane signaling phospholipids in vitro. How exactly CP modulates the properties of filament ends in cells and whether its activity is coordinated by phospholipids in vivo is not well understood. By observing directly the dynamic behavior of individual filament ends in the cortical array of living Arabidopsis thaliana epidermal cells, we dissected the contribution of CP to actin organization and dynamics in response to the signaling phospholipid, phosphatidic acid (PA). Here, we examined three cp knockdown mutants and found that reduced CP levels resulted in more dynamic activity at filament ends, and this significantly enhanced filament-filament annealing and filament elongation from free ends. The cp mutants also exhibited more dense actin filament arrays. Treatment of wild-type cells with exogenous PA phenocopied the actin-based defects in cp mutants, with an increase in the density of filament arrays and enhanced annealing frequency. These cytoskeletal responses to exogenous PA were completely abrogated in cp mutants. Our data provide compelling genetic evidence that the end-capping activity of CP is inhibited by membrane signaling lipids in eukaryotic cells. Specifically, CP acts as a PA biosensor and key transducer of fluxes in membrane signaling phospholipids into changes in actin cytoskeleton dynamics.

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“…The a-subunit gene, CPA (NM_111425 and At3g05520), encodes a polypeptide that is 308 amino acids long and 35,038 D. The b-subunit gene, CPB (NM_105837 and At1g71790), encodes a polypeptide of 256 amino acids and 28,876 D. CP is an obligate heterodimer; for example, genetic ablation of either subunit in budding yeast (S. cerevisiae) leads to loss of the other subunit (Amatruda et al, 1992;Sizonenko et al, 1996;Kim et al, 2004). Similarly, knockdown mutants for either CP subunit in Arabidopsis result in a reduction in transcript levels for the other subunit (Li et al, 2012).…”

Section: Heterodimeric Cp Is a Moderately Abundant Cellular Protein Imentioning

confidence: 99%

Heterodimeric Capping Protein from Arabidopsis Is a Membrane-Associated, Actin-Binding Protein

et al. 2014

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The actin cytoskeleton is a major regulator of cell morphogenesis and responses to biotic and abiotic stimuli. The organization and activities of the cytoskeleton are choreographed by hundreds of accessory proteins. Many actin-binding proteins are thought to be stimulus-response regulators that bind to signaling phospholipids and change their activity upon lipid binding. Whether these proteins associate with and/or are regulated by signaling lipids in plant cells remains poorly understood. Heterodimeric capping protein (CP) is a conserved and ubiquitous regulator of actin dynamics. It binds to the barbed end of filaments with high affinity and modulates filament assembly and disassembly reactions in vitro. Direct interaction of CP with phospholipids, including phosphatidic acid, results in uncapping of filament ends in vitro. Live-cell imaging and reverse-genetic analyses of cp mutants in Arabidopsis (Arabidopsis thaliana) recently provided compelling support for a model in which CP activity is negatively regulated by phosphatidic acid in vivo. Here, we used complementary biochemical, subcellular fractionation, and immunofluorescence microscopy approaches to elucidate CP-membrane association. We found that CP is moderately abundant in Arabidopsis tissues and present in a microsomal membrane fraction. Sucrose density gradient separation and immunoblotting with known compartment markers were used to demonstrate that CP is enriched on membrane-bound organelles such as the endoplasmic reticulum and Golgi. This association could facilitate cross talk between the actin cytoskeleton and a wide spectrum of essential cellular functions such as organelle motility and signal transduction.

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Mutational analysis of capping protein function in Saccharomyces cerevisiae.

Cited by 29 publications

References 32 publications

Arabidopsis Capping Protein (AtCP) Is a Heterodimer That Regulates Assembly at the Barbed Ends of Actin Filaments

Arabidopsis Capping Protein (AtCP) Is a Heterodimer That Regulates Assembly at the Barbed Ends of Actin Filaments

Capping Protein Modulates the Dynamic Behavior of Actin Filaments in Response to Phosphatidic Acid inArabidopsis

Heterodimeric Capping Protein from Arabidopsis Is a Membrane-Associated, Actin-Binding Protein

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