Release of Kinesin from Vesicles by hsc70 and Regulation of Fast Axonal Transport

The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of eitherN-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent andN-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.

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Immunochemical analysis of kinesin light chain function.

Molecular Biology of the Cell ◽

10.1091/mbc.8.4.675 ◽

1997 ◽

Vol 8 (4) ◽

pp. 675-689 ◽

Cited By ~ 96

Author(s):

D L Stenoien ◽

S T Brady

Keyword(s):

Axonal Transport ◽

Light Chain ◽

Membrane Vesicles ◽

Retrograde Transport ◽

Light Chains ◽

Fast Axonal Transport ◽

Kinesin Light Chain ◽

Punctate Pattern ◽

Conventional Kinesin

The kinesin heterotetramer consists of two heavy and two light chains. Kinesin light chains have been proposed to act in binding motor protein to cargo, but evidence for this has been indirect. A library of monoclonal antibodies directed against conserved epitopes throughout the kinesin light chain sequence were used to map light chain functional architecture and to assess physiological functions of these domains. Immunocytochemistry with all antibodies showed a punctate pattern that was detergent soluble. A monoclonal antibody (KLC-All) made against a highly conserved epitope in the tandem repeat domain of light chains inhibited fast axonal transport in isolated axoplasm by decreasing both the number and velocity of vesicles moving, whereas an antibody against a conserved amino terminus epitope had no effect. KLC-All was equally effective at inhibiting both anterograde and retrograde transport. Neither antibody inhibited microtubule-binding or ATPase activity in vitro. KLC-All was unique among antibodies tested in releasing kinesin from purified membrane vesicles, suggesting a mechanism of action for inhibition of axonal transport. These results provide further evidence that conventional kinesin is a motor for fast axonal transport and demonstrate that kinesin light chains play an important role in kinesin interaction with membranes.

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An isoform of kinesin light chain specific for the Golgi complex

Journal of Cell Science ◽

10.1242/jcs.113.11.2047 ◽

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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Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma.

The Journal of Cell Biology ◽

10.1083/jcb.67.3.835 ◽

1975 ◽

Vol 67 (3) ◽

pp. 835-851 ◽

Cited By ~ 1982

Author(s):

G Blobel ◽

B Dobberstein

Keyword(s):

Light Chain ◽

Light Chains ◽

Discussion Section ◽

Heterologous System ◽

In Vitro Processing ◽

Nascent Chain ◽

Detergent Treatment ◽

Membrane Bound

Fractionation of MOPC 41 DL-1 tumors revealed that the mRNA for the light chain of immunoglobulin is localized exclusively in membrane-bound ribosomes. It was shown that the translation product of isolated light chain mRNA in a heterologous protein-synthesizing system in vitro is larger than the authentic secreted light chain; this confirms similar results from several laboratories. The synthesis in vitro of a precursor protein of the light chain is not an artifact of translation in a heterologous system, because it was shown that detached polysomes, isolated from detergent-treated rough microsomes, not only contain nascent light chains which have already been proteolytically processed in vivo but also contain unprocessed nascent light chains. In vitro completion of these nascent light chains thus resulted in the synthesis of some chains having the same mol wt as the authentic secreted light chains, because of completion of in vivo proteolytically processed chains and of other chains which, due to the completion of unprocessed chains, have the same mol wt as the precursor of the light chain. In contrast, completion of the nascent light chains contained in rough microsomes resulted in the synthesis of only processed light chains. Taken together, these results indicate that the processing activity is present in isolated rough microsomes, that it is localized in the membrane moiety of rough microsomes, and, therefore, that it was most likely solubilized during detergent treatment used for the isolation of detached polysomes. Furthermore, these results established that processing in vivo takes place before completion of the nascent chain. The data also indicate that in vitro processing of nascent chains by rough microsomes is dependent on ribosome binding to the membrane. If the latter process is interfered with by aurintricarboxylic acid, rough microsomes also synthesize some unprocessed chains. The data presented in this paper have been interpreted in the light of a recently proposed hypothesis. This hypothesis, referred to as the signal hypothesis, is described in greater detail in the Discussion section.

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Kinesin Light Chains Are Essential for Axonal Transport in Drosophila

The Journal of Cell Biology ◽

10.1083/jcb.141.2.443 ◽

1998 ◽

Vol 141 (2) ◽

pp. 443-454 ◽

Cited By ~ 115

Author(s):

Joseph G. Gindhart ◽

Chand J. Desai ◽

Sven Beushausen ◽

Kai Zinn ◽

Lawrence S.B. Goldstein

Keyword(s):

Axonal Transport ◽

Larval Instar ◽

Cytoplasmic Dynein ◽

Light Chains ◽

Kinesin Light Chain ◽

Heavy Chains ◽

Segmental Nerve ◽

Microtubule Motor ◽

Function Blocks ◽

Mutant Phenotypes

Kinesin is a heterotetramer composed of two 115-kD heavy chains and two 58-kD light chains. The microtubule motor activity of kinesin is performed by the heavy chains, but the functions of the light chains are poorly understood. Mutations were generated in the Drosophila gene Kinesin light chain (Klc), and the phenotypic consequences of loss of Klc function were analyzed at the behavioral and cellular levels. Loss of Klc function results in progressive lethargy, crawling defects, and paralysis followed by death at the end of the second larval instar. Klc mutant axons contain large aggregates of membranous organelles in segmental nerve axons. These aggregates, or organelle jams (Hurd, D.D., and W.M. Saxton. 1996. Genetics. 144: 1075–1085), contain synaptic vesicle precursors as well as organelles that may be transported by kinesin, kinesin-like protein 68D, and cytoplasmic dynein, thus providing evidence that the loss of Klc function blocks multiple pathways of axonal transport. The similarity of the Klc and Khc (Saxton et al. 1991. Cell 64:1093–1102; Hurd, D.D., and W.M. Saxton. 1996. Genetics 144: 1075–1085) mutant phenotypes indicates that KLC is essential for kinesin function, perhaps by tethering KHC to intracellular cargos or by activating the kinesin motor.

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