Identification and partial characterization of mitotic centromere-associated kinesin, a kinesin-related protein that associates with centromeres during mitosis.

Using antipeptide antibodies to conserved regions of the kinesin motor domain, we cloned a kinesin-related protein that associates with the centromere region of mitotic chromosomes. We call the protein MCAK, for mitotic centromere-associated kinesin. MCAK appears concentrated on centromeres at prophase and persists until telophase, after which time the localization disperses. It is found throughout the centromere region and between the kinetochore plates of isolated mitotic CHO chromosomes, in contrast to two other kinetochore-associated microtubule motors: cytoplasmic dynein and CENP-E (Yen et al., 1992), which are closer to the outer surface of the kinetochore plates. Sequence analysis shows MCAK to be a kinesin-related protein with the motor domain located in the center of the protein. It is 60-70% similar to kif2, a kinesin-related protein originally cloned from mouse brain with a centrally located motor domain (Aizawa et al., 1992). MCAK protein is present in interphase and mitotic CHO cells and is transcribed as a single 3.4-kb message.

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Microtubule motors associated with kinetochores in mitotic cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146229 ◽

1993 ◽

Vol 51 ◽

pp. 76-77

Author(s):

Linda Wordeman

Keyword(s):

Mitotic Spindle ◽

Centromere Region ◽

Mitotic Chromosomes ◽

Directed Movement ◽

Culture Cells ◽

Tissue Culture Cells ◽

Microtubule Motors ◽

Double Label ◽

Spindle Microtubules ◽

Kinetochore Region

Chromosomes in dividing tissue culture cells exhibit three types of movement along mitotic spindle microtubules: l)Fast minus-end directed movement (prometaphase), 2)Plus-end directed movement, and 3) Slow minus-end directed movement (anaphase) . In all cases these movements are mediated by the kinetochore region of the centromere of mitotic chromosomes. This region consists of three domains based on both immunocytochemistry and electron microscopy. The outermost or kinetochore domain is composed of the distal fibrous corona and trilamminar plate. The central and pairing domains are located in the chromatin beneath the kinetochore. Both plus- and minus-end directed microtubule motors have been localized to the kinetochore region of mitotic CHO chromosomes. I have used double-label immunocytochemistry to map the location of these motors within the centromere region at the level of the light microscope. Furthermore, I have cloned and expressed a number of novel kinesin-related motors, two of which (Clone 26 and Clone 14) are localized to kinetochores and kinetochore microtubules, respectively.

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Functional Characterization of Borrelia spielmanii Outer Surface Proteins That Interact with Distinct Members of the Human Factor H Protein Family and with Plasminogen

Infection and Immunity ◽

10.1128/iai.00691-09 ◽

2009 ◽

Vol 78 (1) ◽

pp. 39-48 ◽

Cited By ~ 24

Author(s):

Annekatrin Seling ◽

Corinna Siegel ◽

Volker Fingerle ◽

Brandon L. Jutras ◽

Catherine A. Brissette ◽

...

Keyword(s):

Outer Surface ◽

Functional Characterization ◽

Protein Family ◽

Factor H ◽

Surface Proteins ◽

Single Amino Acid ◽

Related Protein ◽

Outer Surface Proteins ◽

Complement Regulator

ABSTRACT Acquisition of complement regulator factor H (CFH) and factor H-like protein 1 (CFHL1) from human serum enables Borrelia spielmanii, one of the etiological agents of Lyme disease, to evade complement-mediated killing by the human host. Up to three distinct complement regulator-acquiring surface proteins (CRASPs) may be expressed by serum-resistant B. spielmanii, each exhibiting an affinity for CFH and/or CFHL1. Here, we describe the functional characterization of the 15-kDa CRASPs of B. spielmanii, members of the polymorphic Erp (OspE/F-related) protein family, that bind two distinct host complement regulators, CFH and factor H-related protein 1 (CFHR1), but not CFHL1. CFH bound to the B. spielmanii CRASPs maintained cofactor activity for factor I-mediated C3b inactivation. Three naturally occurring alleles of this protein bound CFH and CFHR1 while a fourth natural allele could not. Comparative sequence analysis of these protein alleles identified a single amino acid, histidine-79, as playing a significant role in CFH/CFHR1 binding, with substitution by an arginine completely abrogating ligand binding. The mutation of His-79 to Arg did not inhibit binding of plasminogen, another known ligand of this group of borrelial outer-surface proteins.

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Null Mutants of the Neurospora Actin-related Protein 1 Pointed-End Complex Show Distinct Phenotypes

Molecular Biology of the Cell ◽

10.1091/mbc.12.7.2195 ◽

2001 ◽

Vol 12 (7) ◽

pp. 2195-2206 ◽

Cited By ~ 44

Author(s):

In Hyung Lee ◽

Santosh Kumar ◽

Michael Plamann

Keyword(s):

Atpase Activity ◽

Cytoplasmic Dynein ◽

Related Protein ◽

Wild Type ◽

Functional Roles ◽

Distinct Phenotype ◽

High Level ◽

Pointed End

Dynactin is a multisubunit complex that regulates the activities of cytoplasmic dynein, a microtubule-associated motor. Actin-related protein 1 (Arp1) is the most abundant subunit of dynactin, and it forms a short filament to which additional subunits associate. An Arp1 filament pointed-end–binding subcomplex has been identified that consists of p62, p25, p27, and Arp11 subunits. The functional roles of these subunits have not been determined. Recently, we reported the cloning of an apparent homologue of mammalian Arp11 from the filamentous fungus Neurospora crassa. Here, we report that N. crassa ro-2 and ro-12 genes encode the respective p62 and p25 subunits of the pointed-end complex. Characterization of Δro-2, Δro-7, and Δro-12 mutants reveals that each has a distinct phenotype. All three mutants have reduced in vivo vesicle trafficking and have defects in vacuole distribution. We showed previously that in vivo dynactin function is required for high-level dynein ATPase activity, and we find that all three mutants have low dynein ATPase activity. Surprisingly, Δro-12 differs from Δro-2 and Δro-7 and other previously characterized dynein/dynactin mutants in that it has normal nuclear distribution. Each of the mutants shows a distinct dynein/dynactin localization pattern. All three mutants also show stronger dynein/dynactin-membrane interaction relative to wild type, suggesting that the Arp1 pointed-end complex may regulate interaction of dynactin with membranous cargoes.

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Microtubule motors and other maps

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010015753x ◽

1990 ◽

Vol 48 (3) ◽

pp. 1-1

Author(s):

Richard B. Vallee

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Cytoplasmic Dynein ◽

Organelle Transport ◽

Structural Components ◽

Microtubule Associated Proteins ◽

Microtubule Motors ◽

Associated Proteins ◽

The Subject ◽

Intracellular Motility

Microtubules are involved in a number of forms of intracellular motility, including mitosis and bidirectional organelle transport. Purified microtubules from brain and other sources contain tubulin and a diversity of microtubule associated proteins (MAPs). Some of the high molecular weight MAPs - MAP 1A, 1B, 2A, and 2B - are long, fibrous molecules that serve as structural components of the cytamatrix. Three MAPs have recently been identified that show microtubule activated ATPase activity and produce force in association with microtubules. These proteins - kinesin, cytoplasmic dynein, and dynamin - are referred to as cytoplasmic motors. The latter two will be the subject of this talk.Cytoplasmic dynein was first identified as one of the high molecular weight brain MAPs, MAP 1C. It was determined to be structurally equivalent to ciliary and flagellar dynein, and to produce force toward the minus ends of microtubules, opposite to kinesin.

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Faculty Opinions recommendation of Isolation and characterization of a novel rat factor H-related protein that is up-regulated in glomeruli under complement attack.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1010103.143558 ◽

2002 ◽

Author(s):

John Atkinson

Keyword(s):

Factor H ◽

Related Protein ◽

Isolation And Characterization

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Isolation and Characterization of a Cone Snail Protease with Homology to CRISP Proteins of the Pathogenesis-related Protein Superfamily

Journal of Biological Chemistry ◽

10.1074/jbc.m304843200 ◽

2003 ◽

Vol 278 (33) ◽

pp. 31105-31110 ◽

Cited By ~ 155

Author(s):

Trudy J. Milne ◽

Giovanni Abbenante ◽

Joel D. A. Tyndall ◽

Judy Halliday ◽

Richard J. Lewis

Keyword(s):

Cone Snail ◽

Related Protein ◽

Protein Superfamily ◽

Isolation And Characterization ◽

Pathogenesis Related Protein ◽

Pathogenesis Related

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Analysis of the Structural Mechanism of ATP Inhibition at the AAA1 Subunit of Cytoplasmic Dynein-1 Using a Chemical “Toolkit”

International Journal of Molecular Sciences ◽

10.3390/ijms22147704 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7704

Author(s):

Sayi’Mone Tati ◽

Laleh Alisaraie

Keyword(s):

Binding Affinity ◽

Atp Hydrolysis ◽

Critical Role ◽

Motor Protein ◽

Structural Data ◽

Retrograde Transport ◽

Cytoplasmic Dynein ◽

Motor Domain ◽

Conformational Search ◽

Structural Mechanism

Dynein is a ~1.2 MDa cytoskeletal motor protein that carries organelles via retrograde transport in eukaryotic cells. The motor protein belongs to the ATPase family of proteins associated with diverse cellular activities and plays a critical role in transporting cargoes to the minus end of the microtubules. The motor domain of dynein possesses a hexameric head, where ATP hydrolysis occurs. The presented work analyzes the structure–activity relationship (SAR) of dynapyrazole A and B, as well as ciliobrevin A and D, in their various protonated states and their 46 analogues for their binding in the AAA1 subunit, the leading ATP hydrolytic site of the motor domain. This study exploits in silico methods to look at the analogues’ effects on the functionally essential subsites of the motor domain of dynein 1, since no similar experimental structural data are available. Ciliobrevin and its analogues bind to the ATP motifs of the AAA1, namely, the walker-A (W-A) or P-loop, the walker-B (W-B), and the sensor I and II. Ciliobrevin A shows a better binding affinity than its D analogue. Although the double bond in ciliobrevin A and D was expected to decrease the ligand potency, they show a better affinity to the AAA1 binding site than dynapyrazole A and B, lacking the bond. In addition, protonation of the nitrogen atom in ciliobrevin A and D, as well as dynapyrazole A and B, at the N9 site of ciliobrevin and the N7 of the latter increased their binding affinity. Exploring ciliobrevin A geometrical configuration suggests the E isomer has a superior binding profile over the Z due to binding at the critical ATP motifs. Utilizing the refined structure of the motor domain obtained through protein conformational search in this study exhibits that Arg1852 of the yeast cytoplasmic dynein could involve in the “glutamate switch” mechanism in cytoplasmic dynein 1 in lieu of the conserved Asn in AAA+ protein family.

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