scholarly journals Evidence that the stalk of Drosophila kinesin heavy chain is an alpha-helical coiled coil.

1992 ◽  
Vol 116 (4) ◽  
pp. 957-965 ◽  
Author(s):  
M de Cuevas ◽  
T Tao ◽  
L S Goldstein
Keyword(s):  
Circular Dichroism ◽  
Heavy Chain ◽  
Coiled Coil ◽  
Chain Gene ◽  
Tail Domain ◽  
Major Transitions ◽  
Amino Terminal ◽  
Kinesin Heavy Chain ◽  
Stalk Domain ◽  
Circular Dichroïsm

Kinesin is a mechanochemical enzyme composed of three distinct domains: a globular head domain, a rodlike stalk domain, and a small globular tail domain. The stalk domain has sequence features characteristic of alpha-helical coiled coils. To gain insight into the structure of the kinesin stalk, we expressed it from a segment of the Drosophila melanogaster kinesin heavy chain gene and purified it from Escherichia coli. When observed by EM, this protein formed a rodlike structure 40-55 nm long that was occasionally bent at a hingelike region near the middle of the molecule. An additional EM study and a chemical cross-linking study showed that this protein forms a parallel dimer and that the two chains are in register. Finally, using circular dichroism spectroscopy, we showed that this protein is approximately 55-60% alpha-helical in physiological aqueous solution at 25 degrees C, and approximately 85-90% alpha-helical at 4 degrees C. From these results, we conclude that the stalk of kinesin heavy chain forms an alpha-helical coiled coil structure. The temperature dependence of the circular dichroism signal has two major transitions, at 25-30 degrees C and at 45-50 degrees C, which suggests that a portion of the alpha-helical structure in the stalk is less stable than the rest. By producing the amino-terminal (coil 1) and carboxy-terminal (coil 2) halves of the stalk separately in E. coli, we showed that the region that melts below 30 degrees C lies within coil 1, while the majority of coil 2 melts above 45 degrees C. We suggest that this difference in stability may play a role in the force-generating mechanism or regulation of kinesin.

scholarly journals Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells

1992 ◽  
Vol 117 (6) ◽  
pp. 1263-1275 ◽  
Author(s):  
F Navone ◽  
J Niclas ◽  
N Hom-Booher ◽  
L Sparks ◽  
HD Bernstein ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Translational Regulation ◽  
Coiled Coil ◽  
Gene Interaction ◽  
Cloning And Expression ◽  
Upstream Open Reading Frame ◽  
Chain Gene ◽  
Terminal Domain ◽  
Kinesin Heavy Chain ◽  
Cytoplasmic Microtubules

To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOH-terminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesin-like protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with translational regulation in certain mRNAs. After transient expression in CV-1 cells, the kinesin heavy chain showed both a diffuse distribution and a filamentous staining pattern that coaligned with microtubules but not vimentin intermediate filaments. Altering the number and distribution of microtubules with taxol or nocodazole produced corresponding changes in the localization of the expressed kinesin heavy chain. The expressed NH2-terminal motor and the COOH-terminal tail domains, but not the alpha-helical coiled coil rod domain, also colocalized with microtubules. The finding that both the kinesin motor and tail domains can interact with cytoplasmic microtubules raises the possibility that kinesin could crossbridge and induce sliding between microtubules under certain circumstances.

scholarly journals Clonal Tests of Conventional Kinesin Function during Cell Proliferation and Differentiation

2000 ◽  
Vol 11 (4) ◽  
pp. 1329-1343 ◽  
Author(s):  
Robert P. Brendza ◽  
Kathy B. Sheehan ◽  
F.R. Turner ◽  
William M. Saxton
Keyword(s):  
Cell Proliferation ◽  
Endoplasmic Reticulum ◽  
Heavy Chain ◽  
Larval Instar ◽  
Vesicle Transport ◽  
Ultrastructural Changes ◽  
Chain Gene ◽  
Cell Periphery ◽  
Kinesin Heavy Chain ◽  
Conventional Kinesin

Null mutations in the Drosophila Kinesin heavy chain gene (Khc), which are lethal during the second larval instar, have shown that conventional kinesin is critical for fast axonal transport in neurons, but its functions elsewhere are uncertain. To test other tissues, single imaginal cells in young larvae were rendered null for Khc by mitotic recombination. Surprisingly, the null cells produced large clones of adult tissue. The rates of cell proliferation were not reduced, indicating that conventional kinesin is not essential for cell growth or division. This suggests that in undifferentiated cells vesicle transport from the Golgi to either the endoplasmic reticulum or the plasma membrane can proceed at normal rates without conventional kinesin. In adult eye clones produced by null founder cells, there were some defects in differentiation that caused mild ultrastructural changes, but they were not consistent with serious problems in the positioning or transport of endoplasmic reticulum, mitochondria, or vesicles. In contrast, defective cuticle deposition by highly elongated Khc null bristle shafts suggests that conventional kinesin is critical for proper secretory vesicle transport in some cell types, particularly ones that must build and maintain long cytoplasmic extensions. The ubiquity and evolutionary conservation of kinesin heavy chain argue for functions in all cells. We suggest interphase organelle movements away from the cell center are driven by multilayered transport mechanisms; that is, individual organelles can use kinesin-related proteins and myosins, as well as conventional kinesin, to move toward the cell periphery. In this case, other motors can compensate for the loss of conventional kinesin except in cells that have extremely long transport tracks.

scholarly journals Heptad Repeat 2 in Herpes Simplex Virus 1 gH Interacts with Heptad Repeat 1 and Is Critical for Virus Entry and Fusion

2006 ◽  
Vol 80 (5) ◽  
pp. 2216-2224 ◽  
Author(s):  
Tatiana Gianni ◽  
Angela Piccoli ◽  
Carlo Bertucci ◽  
Gabriella Campadelli-Fiume
Keyword(s):  
Circular Dichroism ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Coiled Coil ◽  
Heptad Repeat ◽  
Herpes Simplex Virus 1 ◽  
Mimetic Peptides ◽  
Simplex Virus ◽  
Circular Dichroïsm ◽  
Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) entry into cells and cell-cell fusion mediated by HSV-1 glycoproteins require four glycoproteins, gD, gB, gH, gL. Of these, gH is the only one that so far exhibits structural-functional features typical of viral fusion glycoproteins, i.e., a candidate fusion peptide and, downstream of it, a heptad repeat (HR) segment able to form a coiled coil, named HR-1. Here, we show that gH carries a functional HR-2 capable of physical interaction with HR-1. Specifically, mutational analysis of gH aimed at increasing or decreasing the ability of HR-2 to form a coiled coil resulted in an increase or decrease of fusion activity, respectively. HSV infection was modified accordingly. A mimetic peptide with the HR-2 sequence inhibited HSV-1 infection in a specific and dose-dependent manner. Circular dichroism spectroscopy showed that both HR-2 and HR-1 mimetic peptides adopt mainly random conformation in aqueous solution, while a decrease in peptide environmental polarity determines a conformational change, with a significant increase of the α-helical conformation content, in particular, for the HR-1 peptide. Furthermore, HR-1 and HR-2 mimetic peptides formed a stable complex, as revealed in nondenaturing electrophoresis and by circular dichroism. The mixture of HR-1 and HR-2 peptides reversed the inhibition of HSV infection exerted by the single peptides. Complex formation between HR-1 and HR-2 was independent of the presence of adjacent gH sequences and of additional glycoproteins involved in entry and fusion. Altogether, HR-2 adds to the features typical of class 1 fusion glycoproteins exhibited by HSV-1 gH.

scholarly journals Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport.

1994 ◽  
Vol 127 (4) ◽  
pp. 1041-1048 ◽  
Author(s):  
P A Pesavento ◽  
R J Stewart ◽  
L S Goldstein
Keyword(s):  
Nervous System ◽  
Axonal Transport ◽  
Heavy Chain ◽  
Sequence Similarity ◽  
Coiled Coil ◽  
Biochemical Properties ◽  
Motor Domain ◽  
Tail Region ◽  
Anterograde Axonal Transport ◽  
Kinesin Heavy Chain

This paper describes the molecular and biochemical properties of KLP68D, a new kinesin-like motor protein in Drosophila melanogaster. Sequence analysis of a full-length cDNA encoding KLP68D demonstrates that this protein has a domain that shares significant sequence identity with the entire 340-amin acid kinesin heavy chain motor domain. Sequences extending beyond the motor domain predict a region of alpha-helical coiled-coil followed by a globular "tail" region; there is significant sequence similarity between the alpha-helical coiled-coil region of the KLP68D protein and similar regions of the KIF3 protein of mouse and the KRP85 protein of sea urchin. This finding suggests that all three proteins may be members of the same family, and that they all perform related functions. KLP68D protein produced in Escherichia coli is, like kinesin itself, a plus-end directed microtubule motor. In situ hybridization analysis of KLP68D RNA in Drosophila embryos indicates that the KLP68D gene is expressed primarily in the central nervous system and in a subset of the peripheral nervous system during embryogenesis. Thus, KLP68D may be used for anterograde axonal transport and could conceivably move cargoes in fly neurons different than those moved by kinesin heavy chain or other plus-end directed motors.

scholarly journals Sequence of the clathrin heavy chain from Saccharomyces cerevisiae and requirement of the COOH terminus for clathrin function.

1991 ◽  
Vol 112 (1) ◽  
pp. 65-80 ◽  
Author(s):  
S K Lemmon ◽  
A Pellicena-Palle ◽  
K Conley ◽  
C L Freund
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Heavy Chain ◽  
Coiled Coil ◽  
Coated Vesicle ◽  
Chain Gene ◽  
Temperature Sensitive ◽  
Clathrin Heavy Chain ◽  
And Function ◽  
Major Defect

The sequence of the clathrin heavy chain gene, CHC1, from Saccharomyces cerevisiae is reported. The gene encodes a protein of 1,653 amino acids that is 50% identical to the rat clathrin heavy chain (HC) (Kirchhausen, T., S. C. Harrison, E. P. Chow, R. J. Mattaliano, R. L. Ramachandran, J. Smart, and J. Brosius. 1987. Proc. Natl. Acad. Sci. USA. 84:8805-8809). The alignment extends over the complete length of the two proteins, except for a COOH-terminal extension of the rat HC and a few small gaps, primarily in the globular terminal domain. The yeast HC has four prolines in the region of the rat polypeptide that was proposed to form the binding site for clathrin light chains via an alpha-helical coiled-coil interaction. The yeast protein also lacks the COOH-terminal Pro-Gly rich segment present in the last 45 residues of the rat HC, which were proposed to be involved in the noncovalent association of HCs to form trimers at the triskelion vertex. To examine the importance of the COOH terminus of the HC for clathrin function, a HC containing a COOH-terminal deletion of 57 amino acids (HC delta 57) was expressed in clathrin-deficient yeast (chc1-delta). HC delta 57 rescued some of the phenotypes (slow growth at 30 degrees, genetic instability, and defects in mating and sporulation) associated with the chc1-delta mutation to normal or near normal. Also, truncated HCs were assembled into triskelions. However, cells with HC delta 57 were temperature sensitive for growth and still displayed a major defect in processing of the mating pheromone alpha-factor. Fewer coated vesicles could be isolated from cells with HC delta 57 than cells with the wild-type HC. This suggests that the COOH-terminal region is not required for formation of trimers, but it may be important for normal clathrin-coated vesicle structure and function.

scholarly journals Kinesin- and Myosin-driven Steps of Vesicle Recruitment for Ca2+-regulated Exocytosis

1997 ◽  
Vol 138 (5) ◽  
pp. 999-1008 ◽  
Author(s):  
Guo-Qiang Bi ◽  
Robert L. Morris ◽  
Guochun Liao ◽  
Janet M. Alderton ◽  
Jonathan M. Scholey ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Membrane Trafficking ◽  
Motor Proteins ◽  
Slow Phase ◽  
Cell Periphery ◽  
Tail Domain ◽  
Atpase Inhibitor ◽  
Intact Cells ◽  
Regulated Exocytosis ◽  
Kinesin Heavy Chain

Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca2+-regulated exocytosis by wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin heavy chain specifically inhibited a slow phase, while butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of Ca2+/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast phases, consistent with disruption of a myosin-actin– dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that in intact living cells, kinesin and myosin motors may mediate two sequential transport steps that recruit vesicles to the release sites of Ca2+-regulated exocytosis, although the identity of the responsible myosin isoform is not yet known. They also indicate the existence of three semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of the kinesin heavy chain tail domain.

scholarly journals An unconventional myosin heavy chain gene from Drosophila melanogaster.

1992 ◽  
Vol 119 (4) ◽  
pp. 823-834 ◽  
Author(s):  
K A Kellerman ◽  
K G Miller
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Cytoskeletal Proteins ◽  
Protein Isoforms ◽  
Actin Binding ◽  
Chain Gene ◽  
Cdna Clones ◽  
Western Blots ◽  
Unconventional Myosin ◽  
Amino Terminal

As part of a study of cytoskeletal proteins involved in Drosophila embryonic development, we have undertaken the molecular analysis of a 140-kD ATP-sensitive actin-binding protein (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975). Analysis of cDNA clones encoding this protein revealed that it represents a new class of unconventional myosin heavy chains. The amino-terminal two thirds of the protein comprises a head domain that is 29-33% identical (60-65% similar) to other myosin heads, and contains ATP-binding, actin-binding and calmodulin/myosin light chain-binding motifs. The carboxy-terminal tail has no significant similarity to other known myosin tails, but does contain a approximately 100-amino acid region that is predicted to form an alpha-helical coiled-coil. Since the unique gene that encodes this protein maps to the polytene map position 95F, we have named the new gene Drosophila 95F myosin heavy chain (95F MHC). The expression profile of the 95F MHC gene is complex. Examination of multiple cDNAs reveals that transcripts are alternatively spliced and encode at least three protein isoforms; in addition, a fourth isoform is detected on Western blots. Developmental Northern and Western blots show that transcripts and protein are present throughout the life cycle, with peak expression occurring during mid-embryogenesis and adulthood. Immunolocalization in early embryos demonstrates that the protein is primarily located in a punctate pattern throughout the peripheral cytoplasm. Most cells maintain a low level of protein expression throughout embryogenesis, but specific tissues appear to contain more protein. We speculate that the 95F MHC protein isoforms are involved in multiple dynamic processes during Drosophila development.

A coiled-coil interaction mediates the formation of a ternary complex between dystrophin, dystrobrevin and kinesin heavy chain: Possible implications in the mechanisms of transport

2006 ◽  
Vol 99 (2-3) ◽  
pp. 1 ◽  
Author(s):  
Marina Ceccarini ◽  
Paola Torreri ◽  
Martina Bernassola ◽  
Gianfranco Macchia ◽  
Pompeo Macioce ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Ternary Complex ◽  
Coiled Coil ◽  
Kinesin Heavy Chain

Probing the E/K Peptide Coiled-Coil Assembly by Double Electron–Electron Resonance and Circular Dichroism

Biochemistry ◽  
2020 ◽  
Author(s):  
Elena A. Golysheva ◽  
Aimee L. Boyle ◽  
Barbara Biondi ◽  
Paolo Ruzza ◽  
Alexander Kros ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Coiled Coil ◽  
Electron Resonance ◽  
Double Electron ◽  
Double Electron Electron Resonance ◽  
Circular Dichroïsm
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