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Dictyostelium dynamin B modulates cytoskeletal structures and membranous organelles.

Rai A, Nöthe H, Tzvetkov N, Korenbaum E, Manstein DJ - Cell. Mol. Life Sci. (2010)

Bottom Line: The mature form of dynamin B mediates a wide range and unique combination of functions.The modulating effect of dynamin B on the activity of the contractile vacuole system is unique for the Dictyostelium system.Other functions displayed by dynamin B are commonly associated with either classical dynamins or dynamin-related proteins.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biophysikalische Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Straße, Germany.

ABSTRACT
Dictyostelium discoideum cells produce five dynamin family proteins. Here, we show that dynamin B is the only member of this group of proteins that is initially produced as a preprotein and requires processing by mitochondrial proteases for formation of the mature protein. Our results show that dynamin B-depletion affects many aspects of cell motility, cell-cell and cell-surface adhesion, resistance to osmotic shock, and fatty acid metabolism. The mature form of dynamin B mediates a wide range and unique combination of functions. Dynamin B affects events at the plasma membrane, peroxisomes, the contractile vacuole system, components of the actin-based cytoskeleton, and cell adhesion sites. The modulating effect of dynamin B on the activity of the contractile vacuole system is unique for the Dictyostelium system. Other functions displayed by dynamin B are commonly associated with either classical dynamins or dynamin-related proteins.

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Domain organization and post-translational processing of dynamin B. a Domain organization of dynamin B. Domains are represented as boxes. The numbers indicate the first amino acid residue for each domain; arrow indicates the predicted mitochondrial processing protease cleavage site. b Dynamin B presequence that acts as efficient mitochondrion-targeting sequence (MTS). Positively charged amino acid residues are shown in blue, negatively charged residues in red, hydroxyl-containing residues in green. A predicted R-2 motif is boxed, and the arrow indicates the proteolytic cleavage site. Predicted secondary structure is shown under the sequence. Black rectangles represent α-helices, broken line β-sheets, and straight line unstructured regions. c Molecular weight of native dynamin B. Equal amounts of whole-cell lysates from AX2, AX2 dymB−, and AX2 overproducing ∆NTS-dynamin B cells were separated on 8% SDS-PAGE, blotted, and probed with affinity-purified anti-dynamin B rabbit antibody. d Dynamin B is produced as a longer precursor that is subsequently proteolytically processed. Equal amounts of whole-cell lysates from AX2, dymB− cells, and AX2 cells overproducing dynamin B carrying a His-tag at either the N- or C-terminus were loaded
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Fig1: Domain organization and post-translational processing of dynamin B. a Domain organization of dynamin B. Domains are represented as boxes. The numbers indicate the first amino acid residue for each domain; arrow indicates the predicted mitochondrial processing protease cleavage site. b Dynamin B presequence that acts as efficient mitochondrion-targeting sequence (MTS). Positively charged amino acid residues are shown in blue, negatively charged residues in red, hydroxyl-containing residues in green. A predicted R-2 motif is boxed, and the arrow indicates the proteolytic cleavage site. Predicted secondary structure is shown under the sequence. Black rectangles represent α-helices, broken line β-sheets, and straight line unstructured regions. c Molecular weight of native dynamin B. Equal amounts of whole-cell lysates from AX2, AX2 dymB−, and AX2 overproducing ∆NTS-dynamin B cells were separated on 8% SDS-PAGE, blotted, and probed with affinity-purified anti-dynamin B rabbit antibody. d Dynamin B is produced as a longer precursor that is subsequently proteolytically processed. Equal amounts of whole-cell lysates from AX2, dymB− cells, and AX2 cells overproducing dynamin B carrying a His-tag at either the N- or C-terminus were loaded

Mentions: The coding sequence of dynamin B gene (GenBank AJ251163) consists of 2,760 base pairs and contains no introns. With a calculated molecular mass of 105.3 kDa, dynamin B is one of the largest members of the dynamin family. Dynamin B contains four conserved domains: a consensus GTPase domain, a middle domain, an unstructured QPS domain that is rich in glutamine, proline, and serine residues, and a carboxy-terminal GTPase effector domain (GED) (Fig. 1a). The maximum-likelihood tree constructed by Urushihara and co-workers, which is based on the alignment of 78 dynamin family proteins, indicates that dynamin B is most closely related to Vps1p [31]. In contrast to Vps1p, dynamin B is produced as a preprotein with an unusually long presequence consisting of 136 amino acids. Typically presequences consist of 15–50 amino acids [45]. Presequences serving as mitochondrial targeting sequence are a feature of members of the highly conserved OPA1/Mgm1p family of dynamin-related proteins that control mitochondrial integrity and dynamics [3]. The dynamin B presequence is basic with a pI of 9.8 and rich in Asn (26%), Gln (8%), Ile (10%), Lys (12%), and Tyr (9%) and Ser (8%) residues (Fig. 1b). The GTPase domain of dynamin B (residues 137–516) shows the highest sequence identity for this region with Dictyostelium dynamin A (53%), rat Drp1 (52%), yeast Dnm1p and Vps1p (49%), and human dynamin 1 (48%). The middle domain (residues 517–659) shares the highest degree of sequence identity with dynamin A (32%), Dnm1p (30%), and Drp1 (28%). Residues 660–800 form the QPS-domain. A similar region is present in dynamin A, but not in any other dynamin-like GTPases. It consists of many polar residues (63%), but only a few charged amino acids (12%). This region is especially rich in glutamine (31%), serine (21%), and proline residues (10%), which occur in stretches of up to 12 amino acids. Proline residues are clustered in the center of the QPS domain and may serve a similar function in mediating protein-protein interactions as in the PRD of mammalian dynamin 1. The extreme C-terminus of dynamin B (residues 826–920) shows higher sequence homology to the corresponding domains of other dynamins Drp1 (32%), Dnm1p (30%), dynamin A (29%) and dynamin 1 (21%). This region is known as the GTPase-effector domain (GED) in dynamin 1 because it interacts with the GTPase domain and stimulates its enzymatic activity. Common to GEDs is their strong α-helical character. Based on sequence composition, this is also true for the C-terminal region of dynamin B.Fig. 1


Dictyostelium dynamin B modulates cytoskeletal structures and membranous organelles.

Rai A, Nöthe H, Tzvetkov N, Korenbaum E, Manstein DJ - Cell. Mol. Life Sci. (2010)

Domain organization and post-translational processing of dynamin B. a Domain organization of dynamin B. Domains are represented as boxes. The numbers indicate the first amino acid residue for each domain; arrow indicates the predicted mitochondrial processing protease cleavage site. b Dynamin B presequence that acts as efficient mitochondrion-targeting sequence (MTS). Positively charged amino acid residues are shown in blue, negatively charged residues in red, hydroxyl-containing residues in green. A predicted R-2 motif is boxed, and the arrow indicates the proteolytic cleavage site. Predicted secondary structure is shown under the sequence. Black rectangles represent α-helices, broken line β-sheets, and straight line unstructured regions. c Molecular weight of native dynamin B. Equal amounts of whole-cell lysates from AX2, AX2 dymB−, and AX2 overproducing ∆NTS-dynamin B cells were separated on 8% SDS-PAGE, blotted, and probed with affinity-purified anti-dynamin B rabbit antibody. d Dynamin B is produced as a longer precursor that is subsequently proteolytically processed. Equal amounts of whole-cell lysates from AX2, dymB− cells, and AX2 cells overproducing dynamin B carrying a His-tag at either the N- or C-terminus were loaded
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Fig1: Domain organization and post-translational processing of dynamin B. a Domain organization of dynamin B. Domains are represented as boxes. The numbers indicate the first amino acid residue for each domain; arrow indicates the predicted mitochondrial processing protease cleavage site. b Dynamin B presequence that acts as efficient mitochondrion-targeting sequence (MTS). Positively charged amino acid residues are shown in blue, negatively charged residues in red, hydroxyl-containing residues in green. A predicted R-2 motif is boxed, and the arrow indicates the proteolytic cleavage site. Predicted secondary structure is shown under the sequence. Black rectangles represent α-helices, broken line β-sheets, and straight line unstructured regions. c Molecular weight of native dynamin B. Equal amounts of whole-cell lysates from AX2, AX2 dymB−, and AX2 overproducing ∆NTS-dynamin B cells were separated on 8% SDS-PAGE, blotted, and probed with affinity-purified anti-dynamin B rabbit antibody. d Dynamin B is produced as a longer precursor that is subsequently proteolytically processed. Equal amounts of whole-cell lysates from AX2, dymB− cells, and AX2 cells overproducing dynamin B carrying a His-tag at either the N- or C-terminus were loaded
Mentions: The coding sequence of dynamin B gene (GenBank AJ251163) consists of 2,760 base pairs and contains no introns. With a calculated molecular mass of 105.3 kDa, dynamin B is one of the largest members of the dynamin family. Dynamin B contains four conserved domains: a consensus GTPase domain, a middle domain, an unstructured QPS domain that is rich in glutamine, proline, and serine residues, and a carboxy-terminal GTPase effector domain (GED) (Fig. 1a). The maximum-likelihood tree constructed by Urushihara and co-workers, which is based on the alignment of 78 dynamin family proteins, indicates that dynamin B is most closely related to Vps1p [31]. In contrast to Vps1p, dynamin B is produced as a preprotein with an unusually long presequence consisting of 136 amino acids. Typically presequences consist of 15–50 amino acids [45]. Presequences serving as mitochondrial targeting sequence are a feature of members of the highly conserved OPA1/Mgm1p family of dynamin-related proteins that control mitochondrial integrity and dynamics [3]. The dynamin B presequence is basic with a pI of 9.8 and rich in Asn (26%), Gln (8%), Ile (10%), Lys (12%), and Tyr (9%) and Ser (8%) residues (Fig. 1b). The GTPase domain of dynamin B (residues 137–516) shows the highest sequence identity for this region with Dictyostelium dynamin A (53%), rat Drp1 (52%), yeast Dnm1p and Vps1p (49%), and human dynamin 1 (48%). The middle domain (residues 517–659) shares the highest degree of sequence identity with dynamin A (32%), Dnm1p (30%), and Drp1 (28%). Residues 660–800 form the QPS-domain. A similar region is present in dynamin A, but not in any other dynamin-like GTPases. It consists of many polar residues (63%), but only a few charged amino acids (12%). This region is especially rich in glutamine (31%), serine (21%), and proline residues (10%), which occur in stretches of up to 12 amino acids. Proline residues are clustered in the center of the QPS domain and may serve a similar function in mediating protein-protein interactions as in the PRD of mammalian dynamin 1. The extreme C-terminus of dynamin B (residues 826–920) shows higher sequence homology to the corresponding domains of other dynamins Drp1 (32%), Dnm1p (30%), dynamin A (29%) and dynamin 1 (21%). This region is known as the GTPase-effector domain (GED) in dynamin 1 because it interacts with the GTPase domain and stimulates its enzymatic activity. Common to GEDs is their strong α-helical character. Based on sequence composition, this is also true for the C-terminal region of dynamin B.Fig. 1

Bottom Line: The mature form of dynamin B mediates a wide range and unique combination of functions.The modulating effect of dynamin B on the activity of the contractile vacuole system is unique for the Dictyostelium system.Other functions displayed by dynamin B are commonly associated with either classical dynamins or dynamin-related proteins.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biophysikalische Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Straße, Germany.

ABSTRACT
Dictyostelium discoideum cells produce five dynamin family proteins. Here, we show that dynamin B is the only member of this group of proteins that is initially produced as a preprotein and requires processing by mitochondrial proteases for formation of the mature protein. Our results show that dynamin B-depletion affects many aspects of cell motility, cell-cell and cell-surface adhesion, resistance to osmotic shock, and fatty acid metabolism. The mature form of dynamin B mediates a wide range and unique combination of functions. Dynamin B affects events at the plasma membrane, peroxisomes, the contractile vacuole system, components of the actin-based cytoskeleton, and cell adhesion sites. The modulating effect of dynamin B on the activity of the contractile vacuole system is unique for the Dictyostelium system. Other functions displayed by dynamin B are commonly associated with either classical dynamins or dynamin-related proteins.

Show MeSH
Related in: MedlinePlus