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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 LC deletion mutants with HC. (A) Deletion mutants of LC were constructed in which stop codons were engineered after amino acids 176, 237, 405, and 488. These constructs retain the heptad repeat region but contain deletions of some or all  of the TPR motifs. The deletion mutant LΔHR is missing only the heptad repeat region. Amino acids numbers for the full length protein  are shown below the schematic of LC. (B) COS cells expressing HC, LC, or the LC deletion mutants alone (H, L, L176, L237, L405,  L488, LΔHR; top) or expressing HC together with LC or the LC deletion mutants (H+L, H+L176, H+L237, H+L405, H+L488, and  H+LΔHR; 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 HC and the LC deletion mutants.  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 3: Interaction of the LC deletion mutants with HC. (A) Deletion mutants of LC were constructed in which stop codons were engineered after amino acids 176, 237, 405, and 488. These constructs retain the heptad repeat region but contain deletions of some or all of the TPR motifs. The deletion mutant LΔHR is missing only the heptad repeat region. Amino acids numbers for the full length protein are shown below the schematic of LC. (B) COS cells expressing HC, LC, or the LC deletion mutants alone (H, L, L176, L237, L405, L488, LΔHR; top) or expressing HC together with LC or the LC deletion mutants (H+L, H+L176, H+L237, H+L405, H+L488, and H+LΔHR; 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 HC and the LC deletion mutants. 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: To analyze the effect of LC on the MT binding of HC more directly, a MT cosedimentation assay was developed. COS cells transiently expressing HC with or without LC were lysed with Triton X-100 in a buffer mimicking intracellular conditions. Centrifugation yielded a soluble fraction (lysate) and a pellet containing the insoluble material. Immunoblotting experiments demonstrated that, when expressed alone, ∼30% of the HC remained in the lysate (experimental range 30–50%) (see Fig. 3 B, top, H). LC expressed alone was largely insoluble (see Fig. 3 B, top), in agreement with the observation of LC aggregates by electron microscopy. However, when HC and LC were coexpressed, essentially all of the HC and a significant percentage of LC remained in the lysate (see Fig. 3 B, bottom, H+L). The fraction of LC found in the pellet was variable, depending on the relative levels of expression of the two chains. These results are consistent with HC and LC interacting when coexpressed in COS cells while unassembled LC remains insoluble.


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 LC deletion mutants with HC. (A) Deletion mutants of LC were constructed in which stop codons were engineered after amino acids 176, 237, 405, and 488. These constructs retain the heptad repeat region but contain deletions of some or all  of the TPR motifs. The deletion mutant LΔHR is missing only the heptad repeat region. Amino acids numbers for the full length protein  are shown below the schematic of LC. (B) COS cells expressing HC, LC, or the LC deletion mutants alone (H, L, L176, L237, L405,  L488, LΔHR; top) or expressing HC together with LC or the LC deletion mutants (H+L, H+L176, H+L237, H+L405, H+L488, and  H+LΔHR; 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 HC and the LC deletion mutants.  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 3: Interaction of the LC deletion mutants with HC. (A) Deletion mutants of LC were constructed in which stop codons were engineered after amino acids 176, 237, 405, and 488. These constructs retain the heptad repeat region but contain deletions of some or all of the TPR motifs. The deletion mutant LΔHR is missing only the heptad repeat region. Amino acids numbers for the full length protein are shown below the schematic of LC. (B) COS cells expressing HC, LC, or the LC deletion mutants alone (H, L, L176, L237, L405, L488, LΔHR; top) or expressing HC together with LC or the LC deletion mutants (H+L, H+L176, H+L237, H+L405, H+L488, and H+LΔHR; 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 HC and the LC deletion mutants. 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: To analyze the effect of LC on the MT binding of HC more directly, a MT cosedimentation assay was developed. COS cells transiently expressing HC with or without LC were lysed with Triton X-100 in a buffer mimicking intracellular conditions. Centrifugation yielded a soluble fraction (lysate) and a pellet containing the insoluble material. Immunoblotting experiments demonstrated that, when expressed alone, ∼30% of the HC remained in the lysate (experimental range 30–50%) (see Fig. 3 B, top, H). LC expressed alone was largely insoluble (see Fig. 3 B, top), in agreement with the observation of LC aggregates by electron microscopy. However, when HC and LC were coexpressed, essentially all of the HC and a significant percentage of LC remained in the lysate (see Fig. 3 B, bottom, H+L). The fraction of LC found in the pellet was variable, depending on the relative levels of expression of the two chains. These results are consistent with HC and LC interacting when coexpressed in COS cells while unassembled LC remains insoluble.

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