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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 alone, LC expressed alone, and HC and LC expressed together. COS cells were transiently  transfected with plasmids encoding HC alone (A–D), LC alone (E and F) or both HC and LC (G–J). The expressed proteins were localized by immunofluorescence microscopy. (A and B) HC was detected with an anti–myc monoclonal antibody followed by Rhodamine  Red-X–labeled anti–mouse secondary antibody. (C and D) Cells were double labeled for HC and MTs. HC was detected with anti–myc  polyclonal and Oregon green 488–labeled anti–rabbit antibodies, and tubulin was detected with antitubulin monoclonal and  Rhodamine Red-X–labeled anti–mouse secondary antibodies. (E and F) LC was detected with anti–HA polyclonal and Oregon green  488–labeled anti–rabbit secondary antibodies. (G–J) Cells were double labeled for HC and LC. HC was detected with anti–myc monoclonal and Rhodamine Red-X–labeled anti–mouse antibodies, and LC was detected with anti–HA polyclonal and Oregon green 488– labeled anti–rabbit secondary antibodies. Bar, 10 μm.
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Figure 1: Localization of HC expressed alone, LC expressed alone, and HC and LC expressed together. COS cells were transiently transfected with plasmids encoding HC alone (A–D), LC alone (E and F) or both HC and LC (G–J). The expressed proteins were localized by immunofluorescence microscopy. (A and B) HC was detected with an anti–myc monoclonal antibody followed by Rhodamine Red-X–labeled anti–mouse secondary antibody. (C and D) Cells were double labeled for HC and MTs. HC was detected with anti–myc polyclonal and Oregon green 488–labeled anti–rabbit antibodies, and tubulin was detected with antitubulin monoclonal and Rhodamine Red-X–labeled anti–mouse secondary antibodies. (E and F) LC was detected with anti–HA polyclonal and Oregon green 488–labeled anti–rabbit secondary antibodies. (G–J) Cells were double labeled for HC and LC. HC was detected with anti–myc monoclonal and Rhodamine Red-X–labeled anti–mouse antibodies, and LC was detected with anti–HA polyclonal and Oregon green 488– labeled anti–rabbit secondary antibodies. Bar, 10 μm.

Mentions: Immunofluorescence microscopy of HC expressed alone showed diffuse staining throughout the cytoplasm. A filamentous pattern superimposed upon this diffuse staining was also visible in ∼5–10% of the transfected cells and only in those cells that expressed HC at high levels (Fig. 1, A and B). Double labeling with anti–myc and antitubulin antibodies indicated that the filamentous staining colocalized with MTs (Fig. 1, C and D). In addition, intense staining for HC was often seen at the tips of cellular processes, even when filamentous staining was barely detectable, suggesting that HC is able to move along MTs and accumulate at the plus ends (Fig. 1, C and D). These results confirm previous reports of HC association with MTs upon transient expression in CV-1 (Navone et al., 1992) and mouse L cells (Nakata and Hirokawa, 1995).


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 alone, LC expressed alone, and HC and LC expressed together. COS cells were transiently  transfected with plasmids encoding HC alone (A–D), LC alone (E and F) or both HC and LC (G–J). The expressed proteins were localized by immunofluorescence microscopy. (A and B) HC was detected with an anti–myc monoclonal antibody followed by Rhodamine  Red-X–labeled anti–mouse secondary antibody. (C and D) Cells were double labeled for HC and MTs. HC was detected with anti–myc  polyclonal and Oregon green 488–labeled anti–rabbit antibodies, and tubulin was detected with antitubulin monoclonal and  Rhodamine Red-X–labeled anti–mouse secondary antibodies. (E and F) LC was detected with anti–HA polyclonal and Oregon green  488–labeled anti–rabbit secondary antibodies. (G–J) Cells were double labeled for HC and LC. HC was detected with anti–myc monoclonal and Rhodamine Red-X–labeled anti–mouse antibodies, and LC was detected with anti–HA polyclonal and Oregon green 488– labeled anti–rabbit secondary antibodies. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Localization of HC expressed alone, LC expressed alone, and HC and LC expressed together. COS cells were transiently transfected with plasmids encoding HC alone (A–D), LC alone (E and F) or both HC and LC (G–J). The expressed proteins were localized by immunofluorescence microscopy. (A and B) HC was detected with an anti–myc monoclonal antibody followed by Rhodamine Red-X–labeled anti–mouse secondary antibody. (C and D) Cells were double labeled for HC and MTs. HC was detected with anti–myc polyclonal and Oregon green 488–labeled anti–rabbit antibodies, and tubulin was detected with antitubulin monoclonal and Rhodamine Red-X–labeled anti–mouse secondary antibodies. (E and F) LC was detected with anti–HA polyclonal and Oregon green 488–labeled anti–rabbit secondary antibodies. (G–J) Cells were double labeled for HC and LC. HC was detected with anti–myc monoclonal and Rhodamine Red-X–labeled anti–mouse antibodies, and LC was detected with anti–HA polyclonal and Oregon green 488– labeled anti–rabbit secondary antibodies. Bar, 10 μm.
Mentions: Immunofluorescence microscopy of HC expressed alone showed diffuse staining throughout the cytoplasm. A filamentous pattern superimposed upon this diffuse staining was also visible in ∼5–10% of the transfected cells and only in those cells that expressed HC at high levels (Fig. 1, A and B). Double labeling with anti–myc and antitubulin antibodies indicated that the filamentous staining colocalized with MTs (Fig. 1, C and D). In addition, intense staining for HC was often seen at the tips of cellular processes, even when filamentous staining was barely detectable, suggesting that HC is able to move along MTs and accumulate at the plus ends (Fig. 1, C and D). These results confirm previous reports of HC association with MTs upon transient expression in CV-1 (Navone et al., 1992) and mouse L cells (Nakata and Hirokawa, 1995).

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