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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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Localization of HC expressed with the LC deletion mutants. COS cells were transiently transfected with plasmids encoding  the indicated proteins and the expressed proteins were localized by immunofluorescence microscopy. HC was detected with anti–myc  monoclonal and Rhodamine Red-X–labeled anti–mouse secondary antibodies. The LC deletion mutants were detected with anti–HA  polyclonal and Oregon green 488–labeled anti–rabbit secondary antibodies. For coexpression of HC with L488, note that the cell on the  left expresses both proteins and shows diffuse staining, whereas the cell on the right expresses only HC and shows MT staining (G and  H). Bar, 10 μm.
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Figure 4: Localization of HC expressed with the LC deletion mutants. COS cells were transiently transfected with plasmids encoding the indicated proteins and the expressed proteins were localized by immunofluorescence microscopy. HC was detected with anti–myc monoclonal and Rhodamine Red-X–labeled anti–mouse secondary antibodies. The LC deletion mutants were detected with anti–HA polyclonal and Oregon green 488–labeled anti–rabbit secondary antibodies. For coexpression of HC with L488, note that the cell on the left expresses both proteins and shows diffuse staining, whereas the cell on the right expresses only HC and shows MT staining (G and H). Bar, 10 μm.

Mentions: To determine whether the various LC constructs could inhibit MT binding of HC when they were coexpressed in COS cells, MT binding was first assessed by immunofluorescence microscopy. With the LC constructs lacking one or more TPR motifs, diffuse staining was seen for both the heavy and light chains (Fig. 4, A–H). A filamentous MT-like pattern and accumulation at the tips of processes, characteristic for HC alone, was never seen. However, with the construct lacking the heptad repeat domain (LΔHR), the characteristic MT-staining pattern was still detected for HC, whereas the LC mutant itself showed diffuse staining (Fig. 4, I and J). Similar to full length LC, the mutant LΔHR occasionally showed accumulations in the center of the cell that are likely related to its insolubility (data not shown). The nuclear staining seen for this construct was also variable. These results provide additional evidence that the NH2-terminal domain of LC, containing the heptad repeats, is responsible for the interaction with HC and suggest that this domain is also sufficient to inhibit the MT binding of HC.


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)

Localization of HC expressed with the LC deletion mutants. COS cells were transiently transfected with plasmids encoding  the indicated proteins and the expressed proteins were localized by immunofluorescence microscopy. HC was detected with anti–myc  monoclonal and Rhodamine Red-X–labeled anti–mouse secondary antibodies. The LC deletion mutants were detected with anti–HA  polyclonal and Oregon green 488–labeled anti–rabbit secondary antibodies. For coexpression of HC with L488, note that the cell on the  left expresses both proteins and shows diffuse staining, whereas the cell on the right expresses only HC and shows MT staining (G and  H). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132950&req=5

Figure 4: Localization of HC expressed with the LC deletion mutants. COS cells were transiently transfected with plasmids encoding the indicated proteins and the expressed proteins were localized by immunofluorescence microscopy. HC was detected with anti–myc monoclonal and Rhodamine Red-X–labeled anti–mouse secondary antibodies. The LC deletion mutants were detected with anti–HA polyclonal and Oregon green 488–labeled anti–rabbit secondary antibodies. For coexpression of HC with L488, note that the cell on the left expresses both proteins and shows diffuse staining, whereas the cell on the right expresses only HC and shows MT staining (G and H). Bar, 10 μm.
Mentions: To determine whether the various LC constructs could inhibit MT binding of HC when they were coexpressed in COS cells, MT binding was first assessed by immunofluorescence microscopy. With the LC constructs lacking one or more TPR motifs, diffuse staining was seen for both the heavy and light chains (Fig. 4, A–H). A filamentous MT-like pattern and accumulation at the tips of processes, characteristic for HC alone, was never seen. However, with the construct lacking the heptad repeat domain (LΔHR), the characteristic MT-staining pattern was still detected for HC, whereas the LC mutant itself showed diffuse staining (Fig. 4, I and J). Similar to full length LC, the mutant LΔHR occasionally showed accumulations in the center of the cell that are likely related to its insolubility (data not shown). The nuclear staining seen for this construct was also variable. These results provide additional evidence that the NH2-terminal domain of LC, containing the heptad repeats, is responsible for the interaction with HC and suggest that this domain is also sufficient to inhibit the MT binding of HC.

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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