scholarly journals Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent

2006 ◽  
Vol 173 (4) ◽  
pp. 545-557 ◽  
Author(s):  
Elizabeth E. Glater ◽  
Laura J. Megeath ◽  
R. Steven Stowers ◽  
Thomas L. Schwarz
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Adaptor Protein ◽  
Direct Interaction ◽  
Anterograde Transport ◽  
Kinesin Light Chain ◽  
Energy Demands ◽  
Kinesin Heavy Chain ◽  
Conventional Kinesin ◽  
Dependent Transport

Mitochondria are distributed within cells to match local energy demands. We report that the microtubule-dependent transport of mitochondria depends on the ability of milton to act as an adaptor protein that can recruit the heavy chain of conventional kinesin-1 (kinesin heavy chain [KHC]) to mitochondria. Biochemical and genetic evidence demonstrate that kinesin recruitment and mitochondrial transport are independent of kinesin light chain (KLC); KLC antagonizes milton's association with KHC and is absent from milton–KHC complexes, and mitochondria are present in klc −/− photoreceptor axons. The recruitment of KHC to mitochondria is, in part, determined by the NH2 terminus–splicing variant of milton. A direct interaction occurs between milton and miro, which is a mitochondrial Rho-like GTPase, and this interaction can influence the recruitment of milton to mitochondria. Thus, milton and miro are likely to form an essential protein complex that links KHC to mitochondria for light chain–independent, anterograde transport of mitochondria.

scholarly journals Defective Kinesin Heavy Chain Behavior in Mouse Kinesin Light Chain Mutants

1999 ◽  
Vol 146 (6) ◽  
pp. 1277-1288 ◽  
Author(s):  
Amena Rahman ◽  
Adeela Kamal ◽  
Elizabeth A. Roberts ◽  
Lawrence S.B. Goldstein
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Mutant Mice ◽  
Kinesin Light Chain ◽  
Kinesin Heavy Chain ◽  
Neuronal Tissues ◽  
Biochemical Analyses ◽  
Conventional Kinesin

Conventional kinesin, kinesin-I, is a heterotetramer of two kinesin heavy chain (KHC) subunits (KIF5A, KIF5B, or KIF5C) and two kinesin light chain (KLC) subunits. While KHC contains the motor activity, the role of KLC remains unknown. It has been suggested that KLC is involved in either modulation of KHC activity or in cargo binding. Previously, we characterized KLC genes in mouse (Rahman, A., D.S. Friedman, and L.S. Goldstein. 1998. J. Biol. Chem. 273:15395–15403). Of the two characterized gene products, KLC1 was predominant in neuronal tissues, whereas KLC2 showed a more ubiquitous pattern of expression. To define the in vivo role of KLC, we generated KLC1 gene-targeted mice. Removal of functional KLC1 resulted in significantly smaller mutant mice that also exhibited pronounced motor disabilities. Biochemical analyses demonstrated that KLC1 mutant mice have a pool of KIF5A not associated with any known KLC subunit. Immunofluorescence studies of sensory and motor neuron cell bodies in KLC1 mutants revealed that KIF5A colocalized aberrantly with the peripheral cis-Golgi marker giantin in mutant cells. Striking changes and aberrant colocalization were also observed in the intracellular distribution of KIF5B and β′-COP, a component of COP1 coatomer. Taken together, these data best support models that suggest that KLC1 is essential for proper KHC activation or targeting.

The C-Terminal Region of the Stalk Domain of Ubiquitous Human Kinesin Heavy Chain Contains the Binding Site for Kinesin Light Chain†

Biochemistry ◽  
1998 ◽  
Vol 37 (47) ◽  
pp. 16663-16670 ◽  
Author(s):  
Russell J. Diefenbach ◽  
Joel P. Mackay ◽  
Patricia J. Armati ◽  
Anthony L. Cunningham
Keyword(s):  
Binding Site ◽  
Heavy Chain ◽  
Light Chain ◽  
Terminal Region ◽  
Kinesin Light Chain ◽  
Kinesin Heavy Chain ◽  
Stalk Domain

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 Cytoplasmic Dynein, the Dynactin Complex, and Kinesin Are Interdependent and Essential for Fast Axonal Transport

1999 ◽  
Vol 10 (11) ◽  
pp. 3717-3728 ◽  
Author(s):  
MaryAnn Martin ◽  
Stanley J. Iyadurai ◽  
Andrew Gassman ◽  
Joseph G. Gindhart ◽  
Thomas S. Hays ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Heavy Chain ◽  
Cytoplasmic Dynein ◽  
Genetic Interactions ◽  
Organelle Transport ◽  
Fast Axonal Transport ◽  
Anterograde Transport ◽  
Dynein Heavy Chain ◽  
Conventional Kinesin ◽  
Dynactin Complex

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150Glued(Glued) component of the dynactin complex with the use of genetic techniques in Drosophila.cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150Glued were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued orcDhc64C mutations were stronger than those betweenGlued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.

scholarly journals Light Chain– dependent Regulation of Kinesin's Interaction with Microtubules

1998 ◽  
Vol 143 (4) ◽  
pp. 1053-1066 ◽  
Author(s):  
Kristen J. Verhey ◽  
Donna L. Lizotte ◽  
Tatiana Abramson ◽  
Linda Barenboim ◽  
Bruce J. Schnapp ◽  
...  
Keyword(s):  
Light Chain ◽  
Immunofluorescence Microscopy ◽  
Tail Domain ◽  
Kinesin Light Chain ◽  
Cos Cells ◽  
Sedimentation Behavior ◽  
Cargo Transport ◽  
Stalk Domain ◽  
Heptad Repeats ◽  
Conventional Kinesin

We have investigated the mechanism by which conventional kinesin is prevented from binding to microtubules (MTs) when not transporting cargo. Kinesin heavy chain (HC) was expressed in COS cells either alone or with kinesin light chain (LC). Immunofluorescence microscopy and MT cosedimentation experiments demonstrate that the binding of HC to MTs is inhibited by coexpression of LC. Association between the chains involves the LC NH2-terminal domain, including the heptad repeats, and requires a region of HC that includes the conserved region of the stalk domain and the NH2 terminus of the tail domain. Inhibition of MT binding requires in addition the COOH-terminal 64 amino acids of HC. Interaction between the tail and the motor domains of HC is supported by sedimentation experiments that indicate that kinesin is in a folded conformation. A pH shift from 7.2 to 6.8 releases inhibition of kinesin without changing its sedimentation behavior. Endogenous kinesin in COS cells also shows pH-sensitive inhibition of MT binding. Taken together, our results provide evidence that a function of LC is to keep kinesin in an inactive ground state by inducing an interaction between the tail and motor domains of HC; activation for cargo transport may be triggered by a small conformational change that releases the inhibition of the motor domain for MT binding.

scholarly journals The Structures of Natively Assembled Clathrin Coated Vesicles

2020 ◽  
Author(s):  
Mohammadreza Paraan ◽  
Joshua Mendez ◽  
Savanna Sharum ◽  
Danielle Kurtin ◽  
Huan He ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Associated Factors ◽  
Structural Model ◽  
Protein Complexes ◽  
Vesicle Trafficking ◽  
Adaptor Protein ◽  
Coated Vesicles ◽  
Assembled Complexes ◽  
The Individual

AbstractVesicle trafficking by clathrin coated vesicles (CCVs) is one of the major mechanisms by which proteins and nutrients are absorbed by the cell and transported between organelles. The individual proteins comprising the coated vesicles include clathrin heavy chain, clathrin light chain, and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2-appendages crosslink adjacent CHC β-propellers and that the appendage densities reside almost exclusively in CCV hexagonal faces. We resolve how AP2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.

An isoform of kinesin light chain specific for the Golgi complex

2000 ◽  
Vol 113 (11) ◽  
pp. 2047-2054
Author(s):  
F.K. Gyoeva ◽  
E.M. Bybikova ◽  
A.A. Minin
Keyword(s):  
Golgi Complex ◽  
Light Chain ◽  
Cultured Cells ◽  
Light Chains ◽  
Western Blots ◽  
Kinesin Light Chain ◽  
Heavy Chains ◽  
Carboxyl Terminal ◽  
Kinesin Molecule ◽  
Conventional Kinesin

Conventional kinesin is a motor protein implicated in the transport of a variety of cytoplasmic organelles along microtubules. The kinesin molecule consists of two heavy chains with motor domains at their amino termini and two light chains, which, together with the carboxyl termini of the heavy chains, are proposed to mediate binding to cargoes. Since the light chains are represented by multiple isoforms diverging at their carboxyl termini they are presumed to specify kinesin targeting to organelles. Previously, we isolated five cDNAs, encoding hamster kinesin light chain isoforms, and found that one of them (B or C) preferentially associated with mitochondria. To obtain additional evidence proving the specific location of various kinesin light chain isoforms on organelles, we made an antibody against a 56 amino-acid sequence found at the carboxyl-terminal regions of the hamster D and E isoforms. By indirect immunofluorescence, this antibody specifically labeled the Golgi complex in cultured cells. In western blots of total cell homogenates, it recognized two close polypeptides, one of which co-purified with the Golgi membranes. Thus, the results of this and previous studies demonstrate that different kinesin light chains are associated with different organelles in cells.

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