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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 expressed recombinant  HC+LC and endogenous kinesin is pH dependent. (A) Taxol-stabilized MTs and AMP-PNP  were added to lysates from COS  cells expressing HC (H) or HC  and LC (H+L). Lysates were  prepared in LB+Triton X-100  buffered with MES (pH 6.4),  Pipes (pH 6.8), or Hepes (pH 7.2  and 7.6). MTs and bound proteins were sedimented through a  sucrose cushion and the MT pellets and supernatants were immunoblotted with antibodies  against myc- and HA-epitope  tags to detect the expressed recombinant proteins. Only the  immunoblots of the MT pellets  are shown. (B) The pH dependence of MT binding of HC  alone (circles) or HC and LC  (triangles) was determined between pH 6.8 and 7.2 in LB +  Triton X-100 buffered with  Pipes (solid line) or Hepes  (dashed line). Each data point  represents the mean ± SD of at  least five separate experiments.  (C) Taxol-stabilized MTs and  AMP-PNP were added to lysates, prepared at pH 6.8 or 7.2  as in A, from COS cells coexpressing the indicated recombinant proteins. 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. (D) Sedimentation analysis of coexpressed HC and LC. COS cell lysates made at pH 6.8 (circles) and 7.2 (squares)  were subjected to sedimentation on linear 9–15% sucrose gradients, both at physiological (closed symbols) and high salt (0.5 M NaCl;  open symbols) concentrations. The expressed recombinant proteins were detected in the fractions by immunoblotting with polyclonal  antibodies to the myc- and HA-tags. The mobility on the gradient of proteins with known S values is indicated. (E) Taxol-stabilized MTs  and AMP-PNP were added to lysates, prepared at pH 6.8 or 7.2 as in A, from untransfected COS cells. MTs and bound proteins were  sedimented through a sucrose cushion. The MT pellets (P) and supernatants (S) were immunoblotted to detect endogenous kinesin using a polyclonal antibody to kinesin HC.
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Figure 8: MT cosedimentation of expressed recombinant HC+LC and endogenous kinesin is pH dependent. (A) Taxol-stabilized MTs and AMP-PNP were added to lysates from COS cells expressing HC (H) or HC and LC (H+L). Lysates were prepared in LB+Triton X-100 buffered with MES (pH 6.4), Pipes (pH 6.8), or Hepes (pH 7.2 and 7.6). MTs and bound proteins were sedimented through a sucrose cushion and the MT pellets and supernatants were immunoblotted with antibodies against myc- and HA-epitope tags to detect the expressed recombinant proteins. Only the immunoblots of the MT pellets are shown. (B) The pH dependence of MT binding of HC alone (circles) or HC and LC (triangles) was determined between pH 6.8 and 7.2 in LB + Triton X-100 buffered with Pipes (solid line) or Hepes (dashed line). Each data point represents the mean ± SD of at least five separate experiments. (C) Taxol-stabilized MTs and AMP-PNP were added to lysates, prepared at pH 6.8 or 7.2 as in A, from COS cells coexpressing the indicated recombinant proteins. 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. (D) Sedimentation analysis of coexpressed HC and LC. COS cell lysates made at pH 6.8 (circles) and 7.2 (squares) were subjected to sedimentation on linear 9–15% sucrose gradients, both at physiological (closed symbols) and high salt (0.5 M NaCl; open symbols) concentrations. The expressed recombinant proteins were detected in the fractions by immunoblotting with polyclonal antibodies to the myc- and HA-tags. The mobility on the gradient of proteins with known S values is indicated. (E) Taxol-stabilized MTs and AMP-PNP were added to lysates, prepared at pH 6.8 or 7.2 as in A, from untransfected COS cells. MTs and bound proteins were sedimented through a sucrose cushion. The MT pellets (P) and supernatants (S) were immunoblotted to detect endogenous kinesin using a polyclonal antibody to kinesin HC.

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 expressed recombinant  HC+LC and endogenous kinesin is pH dependent. (A) Taxol-stabilized MTs and AMP-PNP  were added to lysates from COS  cells expressing HC (H) or HC  and LC (H+L). Lysates were  prepared in LB+Triton X-100  buffered with MES (pH 6.4),  Pipes (pH 6.8), or Hepes (pH 7.2  and 7.6). MTs and bound proteins were sedimented through a  sucrose cushion and the MT pellets and supernatants were immunoblotted with antibodies  against myc- and HA-epitope  tags to detect the expressed recombinant proteins. Only the  immunoblots of the MT pellets  are shown. (B) The pH dependence of MT binding of HC  alone (circles) or HC and LC  (triangles) was determined between pH 6.8 and 7.2 in LB +  Triton X-100 buffered with  Pipes (solid line) or Hepes  (dashed line). Each data point  represents the mean ± SD of at  least five separate experiments.  (C) Taxol-stabilized MTs and  AMP-PNP were added to lysates, prepared at pH 6.8 or 7.2  as in A, from COS cells coexpressing the indicated recombinant proteins. 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. (D) Sedimentation analysis of coexpressed HC and LC. COS cell lysates made at pH 6.8 (circles) and 7.2 (squares)  were subjected to sedimentation on linear 9–15% sucrose gradients, both at physiological (closed symbols) and high salt (0.5 M NaCl;  open symbols) concentrations. The expressed recombinant proteins were detected in the fractions by immunoblotting with polyclonal  antibodies to the myc- and HA-tags. The mobility on the gradient of proteins with known S values is indicated. (E) Taxol-stabilized MTs  and AMP-PNP were added to lysates, prepared at pH 6.8 or 7.2 as in A, from untransfected COS cells. MTs and bound proteins were  sedimented through a sucrose cushion. The MT pellets (P) and supernatants (S) were immunoblotted to detect endogenous kinesin using a polyclonal antibody to kinesin HC.
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Figure 8: MT cosedimentation of expressed recombinant HC+LC and endogenous kinesin is pH dependent. (A) Taxol-stabilized MTs and AMP-PNP were added to lysates from COS cells expressing HC (H) or HC and LC (H+L). Lysates were prepared in LB+Triton X-100 buffered with MES (pH 6.4), Pipes (pH 6.8), or Hepes (pH 7.2 and 7.6). MTs and bound proteins were sedimented through a sucrose cushion and the MT pellets and supernatants were immunoblotted with antibodies against myc- and HA-epitope tags to detect the expressed recombinant proteins. Only the immunoblots of the MT pellets are shown. (B) The pH dependence of MT binding of HC alone (circles) or HC and LC (triangles) was determined between pH 6.8 and 7.2 in LB + Triton X-100 buffered with Pipes (solid line) or Hepes (dashed line). Each data point represents the mean ± SD of at least five separate experiments. (C) Taxol-stabilized MTs and AMP-PNP were added to lysates, prepared at pH 6.8 or 7.2 as in A, from COS cells coexpressing the indicated recombinant proteins. 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. (D) Sedimentation analysis of coexpressed HC and LC. COS cell lysates made at pH 6.8 (circles) and 7.2 (squares) were subjected to sedimentation on linear 9–15% sucrose gradients, both at physiological (closed symbols) and high salt (0.5 M NaCl; open symbols) concentrations. The expressed recombinant proteins were detected in the fractions by immunoblotting with polyclonal antibodies to the myc- and HA-tags. The mobility on the gradient of proteins with known S values is indicated. (E) Taxol-stabilized MTs and AMP-PNP were added to lysates, prepared at pH 6.8 or 7.2 as in A, from untransfected COS cells. MTs and bound proteins were sedimented through a sucrose cushion. The MT pellets (P) and supernatants (S) were immunoblotted to detect endogenous kinesin using a polyclonal antibody to kinesin HC.
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.

Show MeSH
Related in: MedlinePlus