Dissection of functional domains by expression of point-mutated profilins in Dictyostelium mutants

Lee, Soo Sim; Karakesisoglou, Iakowos; Noegel, Angelika A.; Rieger, D.; Schleicher, Michael

doi:10.1078/s0171-9335(04)70011-4

Cited by 12 publications

(12 citation statements)

References 59 publications

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“…The GFP-MyoB fusion protein was overexpressed in the myoB null background. Profilin mutants W3N and K114E (Lee et al , 2000) in AX-2 background were maintained in 20 μg/ml hygromycin B (Merck) in HL-5c medium.…”

Section: Methodsmentioning

confidence: 99%

“…The plasmids for the expression of profilins I and II in E. coli are described previously (Lee et al , 2000). The plasmids for Abp1 as a GST or FLAG-tag fusion and the abp1 knockout construct were gifts of Dr. M. de la Roche (Queen's University, Kingston, ON, Canada).…”

Section: Methodsmentioning

confidence: 99%

“…Profilin I and II expression in E. coli and purification were performed as described previously (Kaiser et al , 1989; Lee et al , 2000). …”

Section: Methodsmentioning

confidence: 99%

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A Myosin IK-Abp1-PakB Circuit Acts as a Switch to Regulate Phagocytosis Efficiency

Dieckmann

Heyden

Kistler

et al. 2010

MBoC

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A Myosin IK-Abp1-PakB Circuit Acts as a Switch to Regulate Phagocytosis Efficiency

Dieckmann

Heyden

Kistler

et al. 2010

MBoC

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“…H100E was a minor exception, because a very small fraction of cells depending on this profilin had a cytokinesis defect. Substitutions of these positively charged amino acids in other profilins (human K25Q and K53I; Dictyostelium K114E; and S. cerevisiae R75A, R75G, and R75E) either maintained wildtype PIP 2 binding or complemented profilin null cells (Haarer et al, 1993;Sohn et al, 1995;Lee et al, 2000).…”

Section: Pip 2 Bindingmentioning

confidence: 99%

“…Three are nonconserved amino acids (human H119E, Dictyostelium K114E, and S. cerevisiae H81A Lee et al, 2000;Wolven et al, 2000]). Three (S. cerevisiae S1A, Q3A, D7A; Wolven et al, 2000) are outside the binding sites for known ligands.…”

Section: Other Comparisons With Previous Studiesmentioning

confidence: 99%

Profilin Binding to Poly-l-Proline and Actin Monomers along with Ability to Catalyze Actin Nucleotide Exchange Is Required for Viability of Fission Yeast

Lu¹,

Pollard

2001

MBoC

136

146

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We tested the ability of 87 profilin point mutations to complement temperature-sensitive and null mutations of the single profilin gene of the fission yeast Schizosaccharomyces pombe. We compared the biochemical properties of 13 stable noncomplementing profilins with an equal number of complementing profilin mutants. A large quantitative database revealed the following: 1) in a profilin null background fission yeast grow normally with profilin mutations having Ͼ10% of wild-type affinity for actin or poly-l-proline, but lower affinity for either ligand is incompatible with life; 2) in the cdc3-124 profilin ts background, fission yeast function with profilin having only 2-5% wild-type affinity for actin or poly-l-proline; and 3) special mutations show that the ability of profilin to catalyze nucleotide exchange by actin is an essential function. Thus, poly-l-proline binding, actin binding, and actin nucleotide exchange are each independent requirements for profilin function in fission yeast. INTRODUCTIONProfilins are small proteins that interact with at least three types of ligands: actin monomers (and the homologous Arp2 subunit of Arp2/3 complex); proteins with sequences adopting a type II poly-l-proline helix; and polyphosphoinositides (Machesky and Pollard, 1993;Schluter et al., 1997). Interaction with actin monomers suppresses growth at the pointed end of actin filaments but not the barbed end (Pollard and Cooper, 1984). This allows profilin-actin complexes to support the growth of uncapped barbed ends in cells. In addition, most profilins (amoeba [Mockrin and Korn, 1980;Nishida, 1985], vertebrate [Goldschmidt-Clermont et al., 1991;Perelroizen et al., 1996], yeast [Eads et al., 1998]) but not all profilins (plants [Perelroizen et al., 1996;Eads et al., 1998;Kovar et al., 2000]) stimulate the exchange of the adenine nucleotide bound to actin monomers. This nucleotide exchange activity is postulated to prevent ADP-actin monomers dissociated from filaments from being trapped in highaffinity complexes with ADF/cofilins (Blanchoin and Pollard, 1998;Didry et al., 1998). Potential protein ligands with proline-rich sequences include VASP (Reinhard et al., 1995), Enabled (Ahern-Djamali et al., 1999), Mena (Gertler et al., 1996;Lanier et al., 1999), N-WASP , WAVE/Scar , verprolin/WIP (Ramesh et al., 1997), diaphanous/p140mDia (Watanabe et al., 1997), cappuccino (Manseau et al., 1996), Bni1p and Bnr1p (Evangelista et al., 1997;Imamura et al., 1997), cdc12 (Chang et al., 1997), drebrin and gephyrin (Mammoto et al., 1998), SMN (Giesemann et al., 1999), and aczonin (Wang et al., 1999). In the case of Mena, diaphanous, cappuccino, Bni1p, Bnr1p, and cdc12, genetic interactions support the biological significance of the interaction.Null or conditional mutations established that loss of profilin function is lethal in mice (Lanier et al., 1999), flies (Cooley et al., 1992;Manseau et al., 1996), and fission yeast (Balasubramanian et al., 1994) and severely disabling in Dictyostelium (Haugwitz et al., 1994) and budding yeast (Ha...

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Actin and Actin‐Modulating Proteins

Staiger

Hussey

2018

Annual Plant Reviews Online

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Dissection of functional domains by expression of point-mutated profilins in Dictyostelium mutants

Cited by 12 publications

References 59 publications

A Myosin IK-Abp1-PakB Circuit Acts as a Switch to Regulate Phagocytosis Efficiency

A Myosin IK-Abp1-PakB Circuit Acts as a Switch to Regulate Phagocytosis Efficiency

Profilin Binding to Poly-l-Proline and Actin Monomers along with Ability to Catalyze Actin Nucleotide Exchange Is Required for Viability of Fission Yeast

Actin and Actin‐Modulating Proteins

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