The Regulatory Function Of Amino Acid Region 473-487 Of Prothrombin During Coagulation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3574-3574
Author(s):  
Joesph R Wiencek ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Fold Increase ◽  
The United States ◽  
Regulatory Function ◽  
Wild Type ◽  
Amino Acid Region ◽  
Initial Cleavage ◽  
Acid Region ◽  
Thrombin Formation

Abstract Background In the United States, every thirty nine seconds an individual dies from complications from Cardiovascular Diseases. Persistent thrombus formation at the genesis of these diseases, such as stroke and other coagulation disorders, has no full model to date. Intrinsically blood clots are produced due to excessive/unnecessary thrombin formation, which leads to the conversion of fibrinogen to fibrin. As a result the regulation of thrombin formation is critical in controlling clot generation. Upon vasculature damage, the proteolytic conversion of prothrombin (Pro) to thrombin compatible to rates of survival is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a phospholipid membrane in the presence of calcium ions. Although fXa is capable of activating Pro through the initial cleavage at Arg271 followed by the cleavage at Arg320 (pre2 pathway), it would take approximately six months to form a clot. However, the incorporation of fVa into prothrombinase results in a five-fold increase in the catalytic efficiency of fXa for thrombin generation and the order of cleavages reversed (meizo pathway). Thus the timely arrest of unwarranted bleeding is due to the assembly of prothrombinase at the site of injury. Inevitably the presence and absence of fVa dictates the pathway of Pro activation and previous studies have suggested that fXa interacts with Pro within amino acid region 473-487 in a fVa-dependent manner. Aim To evaluate the role amino acid region 473-487 of Pro has in coagulation. Methods A recombinant Pro molecule with the region 473-487 was deleted (rProΔ473-487) using site-directed mutagenesis. Methotrexate was used for selection to stably transfect BHK-21 cells with rProΔ473-487 and wild-type Pro (rProWT). The two recombinant molecules were purified according to a well-established protocol and, at the last step, Fast Performance Liquid Chromatography was used equipped with a strong anionic Mono-Q 5/50 column. Properly carboxylated rProΔ473-487 and rProWT was isolated and removed from the column by utilizing a calcium gradient. Subsequently Pro deficient plasma was used to assess the molecules clotting activities on a Diagnostica Stago STart® 4 Hemostasis Analyzer. Gel electrophoresis was used to evaluate both recombinant molecules and their ability to generate active thrombin by either the multifaceted prothrombinase or fXa alone. Further studies were then performed using generated recombinant thrombin from the recombinant Pro molecules to investigate in their ability to activate procofactors V (fV) & VIII (fVIII). Results The investigation into the Activated Partial Thromboplastin Time [APTT] revealed clotting activity for human Pro and rProWT to be comparable, whereas rProΔ473-487 was substantially limited in the process of forming a fibrin clot. Next gel electrophoresis and scanning densitometry indicated the consumption of rProΔ473-487 by prothrombinase and subsequent thrombin formation was decreased 24-fold when compared to rProWT. In contrast membrane-bound fXa alone, in the absence of fVa, exhibited a 6-fold increase in the rate of initial cleavage Arg271 and subsequent activation of rProΔ473-487. Both recombinant Pro molecules demonstrated a similar cleavage pattern of activation equivalent with human Pro suggesting no structural alterations took place in rProΔ473-487following the mutation. Furthermore, generated human thrombin and recombinant wild-type thrombin were found to activate fV and fVIII within five minutes while the recombinant mutant thrombin was impeded in the activation process out to three hours. Conclusion Overall the data demonstrate that amino acid sequence 473-487 of Pro plays a preeminent role in 1) timely activation of Pro at initial cleavage Arg320 by prothrombinase, and 2) suitable macromolecular procofactor activation. Thus there is incisive rationale why no major mutations have been identified in this dynamic region which would be problematic for inherent physiological hemostasis. Disclosures: No relevant conflicts of interest to declare.

Role of the N-Terminal Amino Acid Region of Factor Va Light Chain In Prothrombinase Assembly and Function.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1127-1127
Author(s):  
John L. Vaughn ◽  
Jamila Hirbawi ◽  
Michael A. Bukys ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Light Chain ◽  
Gel Electrophoresis ◽  
Fold Increase ◽  
Divalent Ions ◽  
Amino Acid Region ◽  
Factor Va ◽  
And Function ◽  
Acid Region

Abstract Abstract 1127 The timely activation of prothrombin (II) to thrombin (IIa) by the prothrombinase (IIase) complex is required for the maintenance of hemostasis. The IIase complex is composed of the enzyme factor Xa (fXa) and the cofactor factor Va (fVa) assembled on a membrane surface in the presence of divalent ions. While fXa alone is capable of activating II, the incorporation of fVa into the IIase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for IIa formation. FVa is generated following limited proteolysis of factor V (fV) by IIa at Arg709, Arg1018, and Arg1545, releasing a heavy chain (residues 1–709) and light chain (residues 1546–2196) that associate noncovalently through divalent ions. The A3 domain of the light chain of fVa has been implicated in the interaction of the cofactor with fXa. To determine the contribution of the A3 domain of fVa to IIase assembly and function, synthetic peptides representing residues 1546–1612 were screened for their ability to inhibit IIase. The peptide representing residues 1546–1558 strongly inhibited IIase with an IC50 of 50 μM. Additional fluorometric studies with overlapping pentapeptides from that region revealed that amino acids 1553–1558 were responsible for the observed inhibition. To verify the data from the overlapping pentapeptide studies, site-directed mutagenesis was used to generate a fVa mutant molecule with amino acids 1549–1558 deleted (fVaLCD1). FVaLCD1 demonstrated greatly reduced clotting activity in two-stage clotting assays. Gel electrophoresis of the IIase catalyzed activation of II using fVaLCD1 revealed a 50% reduction in the rate of prothrombin consumption. IIase assembled with fVaLCD1 exhibited a 5-fold increase in the apparent disassociation constant (Kdapp) for the enzyme-cofactor interaction and a 50% reduction in the turnover number (kcat) for the enzyme. To investigate the possibility that amino acids 1549–1558 are important for procofactor activation, the fVaLCD1 mutant was incubated with IIa and visualized with gel electrophoresis. The fVaLCD1 mutant exhibited a greatly reduced rate of IIa catalyzed activation. Additional recombinant fVa molecules with overlapping alanine mutations within the amino acid region 1548–1559 revealed that amino acids 1554–1555 were responsible for the observed effects in fVaLCD1. The recombinant fVa molecule with amino acids 1554–1555 mutated to alanines (fVaAA) demonstrated a greatly reduced clotting activity, an 8-fold increase in the Kdapp for the enzyme-cofactor interaction, and a 50% reduction in the kcat for the enzyme. To further investigate the mutational robustness of fVa against mutations in amino acids 1554–1555, a recombinant fVa molecule with both tyrosine residues in that region mutated conservatively to phenylalanines (fVaFF) was created. The more conservative fVaFF mutant, which differed from fVaWT only in the absence of side-chain hydroxyl moieties, exhibited normal clotting activity, kinetics, and procofactor activation. Altogether, the data demonstrate that amino acids 1554–1555 from the N-terminal region of the fVa light chain are important for IIase assembly and function because they constitute an interactive site for IIa and fXa. Disclosures: No relevant conflicts of interest to declare.

Controlling the Pathway for Prothrombin Activation by Prothrombinase.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1695-1695
Author(s):  
Michael A. Bukys ◽  
Paul Y. Kim ◽  
Michael E. Nesheim ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Heavy Chain ◽  
Factor Xa ◽  
Competitive Inhibitor ◽  
Amino Acid Region ◽  
Factor Va ◽  
Initial Cleavage ◽  
Membrane Bound ◽  
Prothrombin Activation ◽  
Thrombin Formation

Abstract Prothrombinase is the enzymatic complex responsible for timely thrombin formation. Activation of human prothrombin is the consequence of two cleavages at Arg271 and Arg320 in prothrombin by factor Xa. Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg271 and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg320 (meizo pathway). We have previously shown that a pentapeptide encompassing amino acid sequence 695–699 from the COOH-terminus of the heavy chain of factor Va (Asp-Tyr-Asp-Tyr-Gln, DYDYQ) interacts with anion binding exosite I (ABE-I) of thrombin and inhibits prothrombin activation by prothrombinase. The peptide was found to be a competitive inhibitor of prothrombinase with respect to substrate. According to the mode of inhibition, we postulated that the peptide binds prothrombin in competition with the binding of the substrate to the enzyme, and inhibits prothrombinase activity by substrate depletion. This mode of DYDYQ inhibition of prothrombin activation by the factor Va-factor Xa complex is similar to that previously demonstrated for sulfated hirugen. To understand the mechanism of inhibition of thrombin formation by DYDYQ we have studied prothrombin activation by gel electrophoresis. Titration of plasma-derived prothrombin activation by fully assembled prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. Higher peptide concentrations were required to impair thrombin formation through the latter pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of prothrombin by prothrombinase at Arg320. These findings were corroborated by studying the kinetics of activation of recombinant mutant prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg320 and Arg271 respectively. Cleavage of rMZ-II by prothrombinase was completely inhibited by low concentrations of DYDYQ while high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with thrombin formation by membrane-bound factor Xa alone in the absence of factor Va. Nonetheless, while the rate for cleavage at Arg271 of plasma-derived prothrombin or rP2-II by membrane-bound factor Xa alone was significantly accelerated in the presence of DYDYQ, resulting in accumulation of prethrombin 2, the rate for cleavage at Arg320 of plasma-derived prothrombin or rMZ-II by membrane-bound factor Xa alone was only moderately affected by the pentapeptide. Our data demonstrate that a pentapeptide mimicking amino acids 695–699 of the heavy chain of factor Va has opposing effects on membrane-bound factor Xa for prothrombin activation, depending on the incorporation of factor Va in prothrombinase. In the presence of the cofactor the peptide inhibits the rate of thrombin generation by specifically interfering with initial cleavage of prothrombin at Arg320, while in the absence of factor Va the pentapeptide accelerates cleavage of prothrombin by factor Xa at Arg271. Thus, the amino acid region spatially surrounding proexosite I in prothrombin most likely has two interactive sites for the components of prothrombinase, a factor Va interactive site and a factor Xa binding site.

Identification of Sulfo-Tyrosines of Factor V.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1731-1731
Author(s):  
Evrim Erdogan ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Factor Xa ◽  
Factor V ◽  
Wild Type ◽  
Single Chain ◽  
Amino Acid Region ◽  
Thrombin Cleavage ◽  
Factor Va ◽  
Cofactor Activity ◽  
Acid Region

Abstract The factor Va molecule is the essential cofactor of the prothrombinase complex. This complex composed of factor Xa and factor Va assembled on a platelet membrane-surface in the presence of Ca2+ ions converts membrane-bound prothrombin to thrombin. Single chain factor V does not bind factor Xa. Single-chain factor V is cleaved by thrombin first at Arg709 followed by cleavages at Arg1018 and Arg1545 to produce the heavy and light chains of the active cofactor (factor Va) and two activation peptides. Efficient thrombin cleavage and activation of factor V is essential for cofactor function and requires tyrosine sulfation. Tyrosine sulfation of factor V also appears to regulate its activity. Seven tyrosine residues in factor V, Tyr665, Tyr696, Tyr698, Tyr1494, Tyr1510, Tyr1515, and Tyr1565 have been identified as potential sites of sulfation. However, which residues are sulfated and their contribution to procofactor activation and cofactor function still remain to be elucidated. Two of the sulfation sites Tyr696 and Tyr698 are located in the acidic amino acid region near to the first required thrombin cleavage site at Arg709. Recent data demonstrated that these residues are essential for factor V activation and cofactor activity. Another acidic amino acid region, 1490–1520 is adjacent to the thrombin cleavage site at Arg1545 required for light chain formation. This region also contains three potential sulfation sites at residues 1494, 1510, and 1515 and was shown to be required for optimum procofactor activation. To ascertain which of these three residues is important for procofactor activation, site-directed mutagenesis was used to create recombinant factor V molecules with mutations 1493DY1494→AF, 1508DDY1510→AAF and 1514DY1515→AF. The clotting and cofactor activity of the 1493DY1494→AF and 1514DY1515→AF mutants was similar to the clotting activity observed with the wild type recombinant factor Va molecule following activation by thrombin or RVV-V activator. In contrast, under similar experimental conditions recombinant factor V with the substitution 1508DDY1510→AAF was deficient in its clotting activity and had impaired cofactor activity. Moreover, following prolonged incubation with thrombin, no light chain formation was observed in the factor V molecule bearing the 1508DDY1510→AAF mutation. Thus, amino acid residues 1508–1510 of factor V are required for thrombin interaction with the procofactor which in turn appears necessary for cleavage at Arg1545. Studies of sulfated proteins have shown that the effect of sulfo-tyrosines on protein structure/function can be preserved by replacing them with glutamic acid. To explicitly identify the sulfated tyrosines on the factor V molecule, we mutated Tyr696, Tyr698 and Tyr1510 to glutamic acid and transfected them into COS-7L cells. Expression was performed in the presence of media containing or devoid of sulfate. In the presence of sulfate, the cofactor and clotting activities of the DY696DY698→DEDE and DDY1510→DDE mutants, separately were similar to the wild type recombinant factor Va molecule. However, in the absence of sulfate, the wild type and the mutant recombinant factor V molecules had both impaired cofactor activity and clotting activity following their activation with thrombin. However, their respective activity was higher than the activity of the factor V molecule bearing the 1508DDY1510→AAF mutation. Our data suggest that residues 696, 698, and 1510 of factor V appear to be sulfated and might be important for procofactor activation and cofactor function.

scholarly journals Stability and dynamics interrelations in a Lipase: Mutational and MD simulations based investigations

2019 ◽  
Author(s):  
Tushar Ranjan Moharana ◽  
Virendra Kumar ◽  
N. Madhusudhana Rao
Keyword(s):  
Amino Acid ◽  
Outer Region ◽  
Md Simulations ◽  
Parameter Analysis ◽  
Amide Bond ◽  
Dynamics Simulation ◽  
Wild Type ◽  
Amino Acid Region ◽  
Non Local ◽  
Acid Region

AbstractDynamics plays crucial role in the function and stability of proteins. Earlier studies have provided ambivalent nature of these interrelations. Epistatic effects of amino acid substitutions on dynamics are an interesting strategy to investigate such relations. In this study we investigated the interrelation between dynamics with that of stability and activity ofBacillus subtilislipase (BSL) using experimental and molecular dynamics simulation (MDS) approaches. Earlier we have identified many stabilising mutations in BSL using directed evolution. In this study these stabilizing mutations were clustered based on their proximity in the sequence into four groups (CM1 to 4). Activity, thermal stability, protease stability and aggregations studies were performed on these four mutants, along with the wild type BSL, to conclude that the mutations in each region contributed additively to the overall stability of the enzyme without suppressing the activity. Root mean square fluctuation and amide bond squared order parameter analysis from MDS revealed that dynamics has increased for CM1, CM2 and CM3 compared to the wild type in the amino acid region 105 to 112 and for CM4 in the amino acid region 22 to 30. In all the mutants core regions dynamics remained unaltered, while the dynamics in the rigid outer region (RMSF <0.05 nm) has increased. Alteration in dynamics, took place both in the vicinity (CM2, 0.41 nm) as well as far away from the mutations (CM1, 2.6 nm; CM3 1.5 nm; CM4 1.7 nm). Our data suggests that enhanced dynamics in certain regions in a protein may actually improve stability.Statement of SignificanceHow does a protein readjust its dynamics upon incorporation of an amino acid that improved its stability? Are the stabilizing effects of a substitution being local or non-local in nature? While there is an excellent documentation (from x-ray studies) of both local and non-local adjustments in interactions upon incorporation of a stabilizing mutations, the effect of these on the protein dynamics is less investigated. The stability and MD data presented here on four mutants, stabilized around four loop regions of a lipase, suggests that stabilizing effects of these mutations influence two specific regions leaving rest of the protein unperturbed. In addition, our data supports, observations by others, wherein enhancement in stability in a protein need not result in dampening of dynamics of a protein.

Wingless signaling generates pattern through two distinct mechanisms

Development ◽  
1997 ◽  
Vol 124 (19) ◽  
pp. 3727-3736 ◽  
Author(s):  
R. Hays ◽  
G.B. Gibori ◽  
A. Bejsovec
Keyword(s):  
Cell Fate ◽  
Cellular Response ◽  
Biological Activities ◽  
Essential Feature ◽  
Structural Features ◽  
Protein Distribution ◽  
Wild Type ◽  
Cell Fates ◽  
Amino Acid Region ◽  
Acid Region

wingless (wg) and its vertebrate homologues, the Wnt genes, play critical roles in the generation of embryonic pattern. In the developing Drosophila epidermis, wg is expressed in a single row of cells in each segment, but it influences cell identities in all rows of epidermal cells in the 10- to 12-cell-wide segment. Wg signaling promotes specification of two distinct aspects of the wild-type intrasegmental pattern: the diversity of denticle types present in the anterior denticle belt and the smooth or naked cuticle constituting the posterior surface of the segment. We have manipulated the expression of wild-type and mutant wg transgenes to explore the mechanism by which a single secreted signaling molecule can promote these distinctly different cell fates. We present evidence consistent with the idea that naked cuticle cell fate is specified by a cellular pathway distinct from the denticle diversity-generating pathway. Since these pathways are differentially activated by mutant Wg ligands, we propose that at least two discrete classes of receptor for Wg may exist, each transducing a different cellular response. We also find that broad Wg protein distribution across many cell diameters is required for the generation of denticle diversity, suggesting that intercellular transport of the Wg protein is an essential feature of pattern formation within the epidermal epithelium. Finally, we demonstrate that an 85 amino acid region not conserved in vertebrate Wnts is dispensable for Wg function and we discuss structural features of the Wingless protein required for its distinct biological activities.

scholarly journals A Carboxy-Terminal 16-Amino-Acid Region of ς38 ofEscherichia coli Is Important for Transcription under High-Salt Conditions and Sigma Activities In Vivo

2000 ◽  
Vol 182 (16) ◽  
pp. 4628-4631 ◽  
Author(s):  
Mio Ohnuma ◽  
Nobuyuki Fujita ◽  
Akira Ishihama ◽  
Kan Tanaka ◽  
Hideo Takahashi
Keyword(s):  
Amino Acid ◽  
Bacterial Species ◽  
High Potassium ◽  
Transcription Analysis ◽  
Amino Acid Region ◽  
Sigma Subunit ◽  
Carboxy Terminal ◽  
Acid Region

ABSTRACT ς38 (or ςS, the rpoS gene product) is a sigma subunit of RNA polymerase in Escherichia coli and directs transcription from a number of stationary-phase promoters as well as osmotically inducible promoters. In this study, we analyzed the function of the carboxy-terminal 16-amino-acid region of ς38 (residues 315 to 330), which is well conserved among the rpoS gene products of enteric bacterial species. Truncation of this region was shown to result in the loss of sigma activity in vivo using promoter-lacZ fusion constructs, but the mutant ς38 retained the binding activity in vivo to the core enzyme. The in vitro transcription analysis revealed that the transcription activity of ς38 holoenzyme under high potassium glutamate concentrations was significantly decreased by the truncation of the carboxy-terminal tail element.

scholarly journals The DNA-binding specificity of the hepatocyte nuclear factor 3/forkhead domain is influenced by amino-acid residues adjacent to the recognition helix.

1994 ◽  
Vol 14 (4) ◽  
pp. 2755-2766 ◽  
Author(s):  
D G Overdier ◽  
A Porcella ◽  
R H Costa
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Hepatocyte Nuclear Factor ◽  
Binding Properties ◽  
Winged Helix ◽  
Amino Acid Region ◽  
Dna Binding Specificity ◽  
Recognition Helix ◽  
Dna Binding Properties ◽  
Acid Region

Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (HNF-3 alpha, -3 beta, and -3 gamma) are known to regulate the transcription of liver-specific genes. The HNF-3 proteins bind to DNA as a monomer through a modified helix-turn-helix, known as the winged helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic forkhead (fkh) protein. We have previously described the isolation, from rodent tissue, of an extensive family of tissue-specific HNF-3/fkh homolog (HFH) genes sharing homology in their winged helix motifs. In this report, we have determined the preferred DNA-binding consensus sequence for the HNF-3 beta protein as well as for two divergent family members, HFH-1 and HFH-2. We show that these HNF-3/fkh proteins bind to distinct DNA sites and that the specificity of protein recognition is dependent on subtle nucleotide alterations in the site. The HNF-3, HFH-1, and HFH-2 consensus binding sequences were also used to search DNA regulatory regions to identify potential target genes. Furthermore, an analysis of the DNA-binding properties of a series of HFH-1/HNF-3 beta protein chimeras has allowed us to identify a 20-amino-acid region, located adjacent to the DNA recognition helix, which contributes to DNA-binding specificity. These sequences are not involved in base-specific contacts and include residues which diverge within the HNF-3/fkh family. Replacement of this 20-amino-acid region in HNF-3 beta with corresponding residues from HFH-1 enabled the HNF-3 beta recognition helix to bind only HFH-1-specific DNA-binding sites. We propose a model in which this 20-amino-acid flanking region influences the DNA-binding properties of the recognition helix.

scholarly journals Rho1p-Bni1p-Spa2p Interactions: Implication in Localization of Bni1p at the Bud Site and Regulation of the Actin Cytoskeleton inSaccharomyces cerevisiae

1998 ◽  
Vol 9 (5) ◽  
pp. 1221-1233 ◽  
Author(s):  
Takeshi Fujiwara ◽  
Kazuma Tanaka ◽  
Akihisa Mino ◽  
Mitsuhiro Kikyo ◽  
Kazuo Takahashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Actin Cytoskeleton ◽  
Binding Protein ◽  
Microscopic Analysis ◽  
Mitogen Activated Protein Kinase ◽  
Gene Encoding ◽  
Amino Acid Region ◽  
Genes Encoding ◽  
Mitogen Activated Protein ◽  
Acid Region

Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213–1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826–987). Genetic analyses revealed that both thebni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.

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