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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 the HC deletion mutants expressed alone or together with LC. Taxol-stabilized MTs and  AMP-PNP were added to lysates from COS cells expressing HC  (H) or the deletion mutants (H682, H810, and H891) alone or together with LC (H+L, H682+L, H810+L, and H891+L). MTs  and bound proteins were sedimented through a sucrose cushion.  The MT pellets (P) and supernatants (S) were immunoblotted to  detect the expressed proteins using polyclonal antibodies to the  myc- and HA-tags.
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Figure 7: MT cosedimentation of the HC deletion mutants expressed alone or together with LC. Taxol-stabilized MTs and AMP-PNP were added to lysates from COS cells expressing HC (H) or the deletion mutants (H682, H810, and H891) alone or together with LC (H+L, H682+L, H810+L, and H891+L). MTs and bound proteins were sedimented through a sucrose cushion. The MT pellets (P) and supernatants (S) were immunoblotted to detect the expressed proteins using polyclonal antibodies to the myc- and HA-tags.

Mentions: To determine whether the same regions of HC that are required for interaction with LC are also involved in the inhibition of MT binding, the HC truncations were expressed alone or together with LC in COS cells. Lysates were subjected to MT cosedimentation assays. When expressed alone, essentially all of the HC truncations sedimented with MTs in the presence of AMP-PNP (Fig. 7, lanes 3, 5, and 7 vs. 11, 13, and 15). When coexpressed with LC, ∼90% of H682 and H810, and at least 50%, but usually much more (experimental range 50–95%), of H891 sedimented with MTs in the presence of AMP-PNP (Fig. 7, lanes 4, 6, and 8 vs. 12, 14, and 16; Fig. 8 C, lanes 11 and 12 vs. 15 and 16). In no case did the expressed HC deletions bind to MTs in the presence of ATP (data not shown). Taken together, the data indicate that although H891 is fully capable of interacting with LC (Fig. 6), this interaction is insufficient to inhibit its MT binding. We therefore conclude that the inhibition of MT binding requires not only an interaction of HC with LC, but also the COOH-terminal 64 amino acids of HC. Similar results were obtained when MT binding was assessed by immunofluorescence microscopy (data not shown).


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 the HC deletion mutants expressed alone or together with LC. Taxol-stabilized MTs and  AMP-PNP were added to lysates from COS cells expressing HC  (H) or the deletion mutants (H682, H810, and H891) alone or together with LC (H+L, H682+L, H810+L, and H891+L). MTs  and bound proteins were sedimented through a sucrose cushion.  The MT pellets (P) and supernatants (S) were immunoblotted to  detect the expressed proteins using polyclonal antibodies to the  myc- and HA-tags.
© Copyright Policy
Related In: Results  -  Collection

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Figure 7: MT cosedimentation of the HC deletion mutants expressed alone or together with LC. Taxol-stabilized MTs and AMP-PNP were added to lysates from COS cells expressing HC (H) or the deletion mutants (H682, H810, and H891) alone or together with LC (H+L, H682+L, H810+L, and H891+L). MTs and bound proteins were sedimented through a sucrose cushion. The MT pellets (P) and supernatants (S) were immunoblotted to detect the expressed proteins using polyclonal antibodies to the myc- and HA-tags.
Mentions: To determine whether the same regions of HC that are required for interaction with LC are also involved in the inhibition of MT binding, the HC truncations were expressed alone or together with LC in COS cells. Lysates were subjected to MT cosedimentation assays. When expressed alone, essentially all of the HC truncations sedimented with MTs in the presence of AMP-PNP (Fig. 7, lanes 3, 5, and 7 vs. 11, 13, and 15). When coexpressed with LC, ∼90% of H682 and H810, and at least 50%, but usually much more (experimental range 50–95%), of H891 sedimented with MTs in the presence of AMP-PNP (Fig. 7, lanes 4, 6, and 8 vs. 12, 14, and 16; Fig. 8 C, lanes 11 and 12 vs. 15 and 16). In no case did the expressed HC deletions bind to MTs in the presence of ATP (data not shown). Taken together, the data indicate that although H891 is fully capable of interacting with LC (Fig. 6), this interaction is insufficient to inhibit its MT binding. We therefore conclude that the inhibition of MT binding requires not only an interaction of HC with LC, but also the COOH-terminal 64 amino acids of HC. Similar results were obtained when MT binding was assessed by immunofluorescence microscopy (data not shown).

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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