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Light chain-dependent regulation of Kinesin's interaction with microtubules.

Verhey KJ, Lizotte DL, Abramson T, Barenboim L, Schnapp BJ, Rapoport TA - J. Cell Biol. (1998)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
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.

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MT cosedimentation of HC expressed alone or together with LC. (A) Lysates from COS cells expressing HC alone  (H) or together with LC (H+L) were compared under three conditions: no addition of MTs (left) or addition of taxol-stabilized  MTs with either AMP-PNP (middle) or ATP (right). MTs and  bound proteins were sedimented through a sucrose cushion and  the MT pellets (P) and supernatants (S) were immunoblotted for  the presence of the expressed HC and LC. (B) Quantitation  of the amount of HC recovered in the MT pellet in the presence  of AMP-PNP as a function of the amount of MTs added to the lysate. (•) Data from cells expressing HC alone, (▴) cells coexpressing HC and LC. Each data point represents the mean ± SD  of at least four experiments.
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Figure 2: MT cosedimentation of HC expressed alone or together with LC. (A) Lysates from COS cells expressing HC alone (H) or together with LC (H+L) were compared under three conditions: no addition of MTs (left) or addition of taxol-stabilized MTs with either AMP-PNP (middle) or ATP (right). MTs and bound proteins were sedimented through a sucrose cushion and the MT pellets (P) and supernatants (S) were immunoblotted for the presence of the expressed HC and LC. (B) Quantitation of the amount of HC recovered in the MT pellet in the presence of AMP-PNP as a function of the amount of MTs added to the lysate. (•) Data from cells expressing HC alone, (▴) cells coexpressing HC and LC. Each data point represents the mean ± SD of at least four experiments.

Mentions: Next, taxol-stabilized MTs were added to the lysates from cells expressing either HC alone or both HC and LC. Incubations were carried out in the presence of AMP-PNP or ATP, the MTs were sedimented, and the amounts of HC and LC in the pellet were determined by immunoblotting. When expressed alone, the majority of HC cosedimented with MTs in the presence of AMP-PNP (Fig. 2 A, lane 5 vs. 7), but remained in the supernatant in the presence of ATP (lane 9 vs. 11). In contrast, most of the HC failed to bind to MTs when coexpressed with LC, even in the presence of AMP-PNP (Fig. 2 A, lane 6 vs. 8), although some binding was observed when very high concentrations of MTs were added (Fig. 2 B). These results confirm the conclusion drawn from the immunofluorescence experiments and show directly that the interaction of LC with HC reduces the ability of HC to bind microtubules.


Light chain-dependent regulation of Kinesin's interaction with microtubules.

Verhey KJ, Lizotte DL, Abramson T, Barenboim L, Schnapp BJ, Rapoport TA - J. Cell Biol. (1998)

MT cosedimentation of HC expressed alone or together with LC. (A) Lysates from COS cells expressing HC alone  (H) or together with LC (H+L) were compared under three conditions: no addition of MTs (left) or addition of taxol-stabilized  MTs with either AMP-PNP (middle) or ATP (right). MTs and  bound proteins were sedimented through a sucrose cushion and  the MT pellets (P) and supernatants (S) were immunoblotted for  the presence of the expressed HC and LC. (B) Quantitation  of the amount of HC recovered in the MT pellet in the presence  of AMP-PNP as a function of the amount of MTs added to the lysate. (•) Data from cells expressing HC alone, (▴) cells coexpressing HC and LC. Each data point represents the mean ± SD  of at least four experiments.
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Related In: Results  -  Collection

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Figure 2: MT cosedimentation of HC expressed alone or together with LC. (A) Lysates from COS cells expressing HC alone (H) or together with LC (H+L) were compared under three conditions: no addition of MTs (left) or addition of taxol-stabilized MTs with either AMP-PNP (middle) or ATP (right). MTs and bound proteins were sedimented through a sucrose cushion and the MT pellets (P) and supernatants (S) were immunoblotted for the presence of the expressed HC and LC. (B) Quantitation of the amount of HC recovered in the MT pellet in the presence of AMP-PNP as a function of the amount of MTs added to the lysate. (•) Data from cells expressing HC alone, (▴) cells coexpressing HC and LC. Each data point represents the mean ± SD of at least four experiments.
Mentions: Next, taxol-stabilized MTs were added to the lysates from cells expressing either HC alone or both HC and LC. Incubations were carried out in the presence of AMP-PNP or ATP, the MTs were sedimented, and the amounts of HC and LC in the pellet were determined by immunoblotting. When expressed alone, the majority of HC cosedimented with MTs in the presence of AMP-PNP (Fig. 2 A, lane 5 vs. 7), but remained in the supernatant in the presence of ATP (lane 9 vs. 11). In contrast, most of the HC failed to bind to MTs when coexpressed with LC, even in the presence of AMP-PNP (Fig. 2 A, lane 6 vs. 8), although some binding was observed when very high concentrations of MTs were added (Fig. 2 B). These results confirm the conclusion drawn from the immunofluorescence experiments and show directly that the interaction of LC with HC reduces the ability of HC to bind microtubules.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus