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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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Interaction of the HC deletion mutants with LC. (A) Deletion mutants of HC were constructed in which stop codons were engineered after amino acids 682, 810, and 891. These deletions remove both the tail domain and the conserved region of the stalk domain  (H682), the entire tail domain (H810), or half of the tail domain (H891). (B) COS cells expressing HC or the HC deletion mutants alone  (H, H682, H810, and H891; top) or together with LC (H+L, H682+L, H810+L, and H891+L; bottom) were lysed with 1% Triton  X-100. After centrifugation, the amount of expressed protein found insoluble in the pellet (P) and soluble in the lysate (S) was determined by immunoblotting with polyclonal antibodies to the myc- and HA-epitope tags. Molecular weight size standards (kilodaltons)  are indicated on the left. (C) Coimmunoprecipitation of the HC deletion mutants and LC. Lysates were immunoprecipitated for HC using a monoclonal anti–myc antibody (left) or for LC using a monoclonal anti–HA antibody (right). Immunoprecipitates were immunoblotted to detect the expressed proteins using polyclonal antibodies to the epitope tags.
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Figure 6: Interaction of the HC deletion mutants with LC. (A) Deletion mutants of HC were constructed in which stop codons were engineered after amino acids 682, 810, and 891. These deletions remove both the tail domain and the conserved region of the stalk domain (H682), the entire tail domain (H810), or half of the tail domain (H891). (B) COS cells expressing HC or the HC deletion mutants alone (H, H682, H810, and H891; top) or together with LC (H+L, H682+L, H810+L, and H891+L; bottom) were lysed with 1% Triton X-100. After centrifugation, the amount of expressed protein found insoluble in the pellet (P) and soluble in the lysate (S) was determined by immunoblotting with polyclonal antibodies to the myc- and HA-epitope tags. Molecular weight size standards (kilodaltons) are indicated on the left. (C) Coimmunoprecipitation of the HC deletion mutants and LC. Lysates were immunoprecipitated for HC using a monoclonal anti–myc antibody (left) or for LC using a monoclonal anti–HA antibody (right). Immunoprecipitates were immunoblotted to detect the expressed proteins using polyclonal antibodies to the epitope tags.

Mentions: A similar approach was used to determine the region of HC required for interaction with LC. Truncation mutants were made that either lacked the tail domain and the COOH-terminal portion of the stalk domain (H682), just the tail domain (H810), or the COOH-terminal half of the tail domain (H891) (Fig. 6 A). The proteins were expressed in COS cells alone or together with LC and lysates were analyzed by immunoblotting. In each case, a protein product of the predicted size was detected (Fig. 6 B). Whether expressed alone or together with LC, the expressed H682 and H810 were largely in the supernatant after centrifugation (Fig. 6 B, top). On the other hand, similar to full length HC, much of H891 was in the insoluble pellet when expressed alone, but was rendered soluble when coexpressed with LC. Nearly all of LC was insoluble when coexpressed with HC lacking the tail domain and some of the stalk (H682) (Fig. 6 B, bottom). In contrast, a significant portion of LC was in the supernatant when coexpressed with the longer HC constructs (H810 or H891). These data suggest that the COOH-terminal region of the HC stalk domain is required for the interaction with LC.


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)

Interaction of the HC deletion mutants with LC. (A) Deletion mutants of HC were constructed in which stop codons were engineered after amino acids 682, 810, and 891. These deletions remove both the tail domain and the conserved region of the stalk domain  (H682), the entire tail domain (H810), or half of the tail domain (H891). (B) COS cells expressing HC or the HC deletion mutants alone  (H, H682, H810, and H891; top) or together with LC (H+L, H682+L, H810+L, and H891+L; bottom) were lysed with 1% Triton  X-100. After centrifugation, the amount of expressed protein found insoluble in the pellet (P) and soluble in the lysate (S) was determined by immunoblotting with polyclonal antibodies to the myc- and HA-epitope tags. Molecular weight size standards (kilodaltons)  are indicated on the left. (C) Coimmunoprecipitation of the HC deletion mutants and LC. Lysates were immunoprecipitated for HC using a monoclonal anti–myc antibody (left) or for LC using a monoclonal anti–HA antibody (right). Immunoprecipitates were immunoblotted to detect the expressed proteins using polyclonal antibodies to the epitope tags.
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Related In: Results  -  Collection

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Figure 6: Interaction of the HC deletion mutants with LC. (A) Deletion mutants of HC were constructed in which stop codons were engineered after amino acids 682, 810, and 891. These deletions remove both the tail domain and the conserved region of the stalk domain (H682), the entire tail domain (H810), or half of the tail domain (H891). (B) COS cells expressing HC or the HC deletion mutants alone (H, H682, H810, and H891; top) or together with LC (H+L, H682+L, H810+L, and H891+L; bottom) were lysed with 1% Triton X-100. After centrifugation, the amount of expressed protein found insoluble in the pellet (P) and soluble in the lysate (S) was determined by immunoblotting with polyclonal antibodies to the myc- and HA-epitope tags. Molecular weight size standards (kilodaltons) are indicated on the left. (C) Coimmunoprecipitation of the HC deletion mutants and LC. Lysates were immunoprecipitated for HC using a monoclonal anti–myc antibody (left) or for LC using a monoclonal anti–HA antibody (right). Immunoprecipitates were immunoblotted to detect the expressed proteins using polyclonal antibodies to the epitope tags.
Mentions: A similar approach was used to determine the region of HC required for interaction with LC. Truncation mutants were made that either lacked the tail domain and the COOH-terminal portion of the stalk domain (H682), just the tail domain (H810), or the COOH-terminal half of the tail domain (H891) (Fig. 6 A). The proteins were expressed in COS cells alone or together with LC and lysates were analyzed by immunoblotting. In each case, a protein product of the predicted size was detected (Fig. 6 B). Whether expressed alone or together with LC, the expressed H682 and H810 were largely in the supernatant after centrifugation (Fig. 6 B, top). On the other hand, similar to full length HC, much of H891 was in the insoluble pellet when expressed alone, but was rendered soluble when coexpressed with LC. Nearly all of LC was insoluble when coexpressed with HC lacking the tail domain and some of the stalk (H682) (Fig. 6 B, bottom). In contrast, a significant portion of LC was in the supernatant when coexpressed with the longer HC constructs (H810 or H891). These data suggest that the COOH-terminal region of the HC stalk domain is required for the interaction with LC.

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