Determinants of kinesin motor polarity

Science

Volumn 281, Issue 5380, 1998, Pages 1200-1202

  Endow, Sharyn A ^a   Waligora, Kimberly W ^a  

^a DUKE UNIVERSITY MEDICAL CENTER   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CHIMERIC PROTEIN; KINESIN;

ARTICLE; CONTROLLED STUDY; DROSOPHILA MELANOGASTER; MICROTUBULE; MOVEMENT (PHYSIOLOGY); NONHUMAN; POLARIZATION; PRIORITY JOURNAL;

DROSOPHILA MELANOGASTER; MELANOGASTER;

EID: 0032555532     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.281.5380.1200     Document Type: Article

References (28)

1. G. S. Bloom and S. A. Endow, Protein Profile 2, 1109 (1995); R. D. Vale. J. Cell Biol. 135, 291 (1996); N. Hirokawa, Science 279, 519 (1998).
2. G. S. Bloom and S. A. Endow, Protein Profile 2, 1109 (1995); R. D. Vale. J. Cell Biol. 135, 291 (1996); N. Hirokawa, Science 279, 519 (1998).
3. G. S. Bloom and S. A. Endow, Protein Profile 2, 1109 (1995); R. D. Vale. J. Cell Biol. 135, 291 (1996); N. Hirokawa, Science 279, 519 (1998).
4. U. Henningsen and M. Schliwa, Nature 389, 93 (1997).
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6. E. P. Sablin, F. J. Kull, R. Cooke, R. D. Vale, R. J. Fletterick, Nature 380, 555 (1996); F. J. Kull, E. P. Sablin, R. Lau, R. J. Fletterick, R. D. Vale, ibid., p. 550.
7. E. P. Sablin, F. J. Kull, R. Cooke, R. D. Vale, R. J. Fletterick, Nature 380, 555 (1996); F. J. Kull, E. P. Sablin, R. Lau, R. J. Fletterick, R. D. Vale, ibid., p. 550.
8. S. A. Endow and R. J. Fletterick, Bioessays 20, 108 (1998).
9. A. Lupas and M. Van Dyke, J. Stock, Science 252, 1162 (1991).
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11. I. M.-T. C. Crevel, A. Lockhart, R. A. Cross, J. Mol. Biol. 273, 160 (1997); R. D. Vale and R. J. Fletterick, Annu. Rev. Cell Dev. Biol. 13, 745 (1997); L. Romberg, D. W. Pierce, R. D. Vale, J. Cell Biol. 140, 1407 (1998).
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13. F. Kozielski et al., Cell 91, 985 (1997).
14. NcdKHC1 consists of glutathione S-transferase (GST) fused to the Ncd stalk and neck followed by the Drosophila KHC motor core and the Ncd COOH-terminus [residues 664 to 700 that extend beyond the conserved motor core and may interact with the Ncd neck (4)]. NcdKHC2-5 are mutants of NcdKHC1 (Fig. 1). NcdKHC6 consists of the Ned stalk, neck, and motor with the Ncd COOH-terminus replaced by the Drosophila KHC neck.
15. The pGEX/ncdkhc1-5 chimeric plasmids were constructed from a plasmid coding for GST fused to Ncd residues 194 to 346 [S. A. Endow, S. Henikoff, L. Soler-Niedziela, Nature 345, 81 (1990)], followed by Drosophila KHC residues 11 to 340 [J. T. Yang, R. A. Laymon, L S. B. Goldstein, Cell 56, 879 (1989)]. The plasmid was constructed by using the polymerase chain reaction (PCR) and wild-type ncd and khc plasmids [H. Song and S. A. Endow, Biochemistry 35, 11203 (1996)]. A Bam HI site was created to ligate ncd to pGEX-2T [D. B. Smith and K. S. Johnson, Gene 67, 31 (1988)] and a Bss HII site was created for the junction with khc, chosen because it minimally altered the Ncd neck according to the computer program COILS (6), changing L345A. The khc primers contained a Bss HII site, sequences coding for residues 338 to 340 missing from the khc plasmid, and an Eco RI site for ligation to pGEX-2T. pGEX/ncdkhc2 and -3 were made by replacing the Nsi I-Eco RI fragment of the plasmid with overlap extension PCR fragments [S. N. Ho, H. D. Hunt, R. M. Horton, J. K. Pullen, L. R. Pease, Gene 77, 51 (1989)] to substitute Ncd residues 664 to 700 for KHC residues 327 to 340 or to delete KHC after residue 326 and add a glycine. pGEX/ncdkhc1, -4, and -5 were made from pGEX/ncdkhc2 by replacing the Sph I-Nsi I fragment with overlap PCR fragments that repaired the L345A mutation in the Ncd neck or shifted the neck-motor junction by two residues or both. pGCX/ncdkhc6 was made from pGEX/MC1 (73) and wild-type khc by replacing the pGEX/MC1 Bst EII-Eco RI fragment with an overlap PCR fragment coding for Ncd to residue 663, followed by KHC residues 327 to 340. All plasmids were sequenced to confirm the intended changes and exclude PCR mutations.
16. The pGEX/ncdkhc1-5 chimeric plasmids were constructed from a plasmid coding for GST fused to Ncd residues 194 to 346 [S. A. Endow, S. Henikoff, L. Soler-Niedziela, Nature 345, 81 (1990)], followed by Drosophila KHC residues 11 to 340 [J. T. Yang, R. A. Laymon, L S. B. Goldstein, Cell 56, 879 (1989)]. The plasmid was constructed by using the polymerase chain reaction (PCR) and wild-type ncd and khc plasmids [H. Song and S. A. Endow, Biochemistry 35, 11203 (1996)]. A Bam HI site was created to ligate ncd to pGEX-2T [D. B. Smith and K. S. Johnson, Gene 67, 31 (1988)] and a Bss HII site was created for the junction with khc, chosen because it minimally altered the Ncd neck according to the computer program COILS (6), changing L345A. The khc primers contained a Bss HII site, sequences coding for residues 338 to 340 missing from the khc plasmid, and an Eco RI site for ligation to pGEX-2T. pGEX/ncdkhc2 and -3 were made by replacing the Nsi I-Eco RI fragment of the plasmid with overlap extension PCR fragments [S. N. Ho, H. D. Hunt, R. M. Horton, J. K. Pullen, L. R. Pease, Gene 77, 51 (1989)] to substitute Ncd residues 664 to 700 for KHC residues 327 to 340 or to delete KHC after residue 326 and add a glycine. pGEX/ncdkhc1, -4, and -5 were made from pGEX/ncdkhc2 by replacing the Sph I-Nsi I fragment with overlap PCR fragments that repaired the L345A mutation in the Ncd neck or shifted the neck-motor junction by two residues or both. pGCX/ncdkhc6 was made from pGEX/MC1 (73) and wild-type khc by replacing the pGEX/MC1 Bst EII-Eco RI fragment with an overlap PCR fragment coding for Ncd to residue 663, followed by KHC residues 327 to 340. All plasmids were sequenced to confirm the intended changes and exclude PCR mutations.
17. The pGEX/ncdkhc1-5 chimeric plasmids were constructed from a plasmid coding for GST fused to Ncd residues 194 to 346 [S. A. Endow, S. Henikoff, L. Soler-Niedziela, Nature 345, 81 (1990)], followed by Drosophila KHC residues 11 to 340 [J. T. Yang, R. A. Laymon, L S. B. Goldstein, Cell 56, 879 (1989)]. The plasmid was constructed by using the polymerase chain reaction (PCR) and wild-type ncd and khc plasmids [H. Song and S. A. Endow, Biochemistry 35, 11203 (1996)]. A Bam HI site was created to ligate ncd to pGEX-2T [D. B. Smith and K. S. Johnson, Gene 67, 31 (1988)] and a Bss HII site was created for the junction with khc, chosen because it minimally altered the Ncd neck according to the computer program COILS (6), changing L345A. The khc primers contained a Bss HII site, sequences coding for residues 338 to 340 missing from the khc plasmid, and an Eco RI site for ligation to pGEX-2T. pGEX/ncdkhc2 and -3 were made by replacing the Nsi I-Eco RI fragment of the plasmid with overlap extension PCR fragments [S. N. Ho, H. D. Hunt, R. M. Horton, J. K. Pullen, L. R. Pease, Gene 77, 51 (1989)] to substitute Ncd residues 664 to 700 for KHC residues 327 to 340 or to delete KHC after residue 326 and add a glycine. pGEX/ncdkhc1, -4, and -5 were made from pGEX/ncdkhc2 by replacing the Sph I-Nsi I fragment with overlap PCR fragments that repaired the L345A mutation in the Ncd neck or shifted the neck-motor junction by two residues or both. pGCX/ncdkhc6 was made from pGEX/MC1 (73) and wild-type khc by replacing the pGEX/MC1 Bst EII-Eco RI fragment with an overlap PCR fragment coding for Ncd to residue 663, followed by KHC residues 327 to 340. All plasmids were sequenced to confirm the intended changes and exclude PCR mutations.
18. The pGEX/ncdkhc1-5 chimeric plasmids were constructed from a plasmid coding for GST fused to Ncd residues 194 to 346 [S. A. Endow, S. Henikoff, L. Soler-Niedziela, Nature 345, 81 (1990)], followed by Drosophila KHC residues 11 to 340 [J. T. Yang, R. A. Laymon, L S. B. Goldstein, Cell 56, 879 (1989)]. The plasmid was constructed by using the polymerase chain reaction (PCR) and wild-type ncd and khc plasmids [H. Song and S. A. Endow, Biochemistry 35, 11203 (1996)]. A Bam HI site was created to ligate ncd to pGEX-2T [D. B. Smith and K. S. Johnson, Gene 67, 31 (1988)] and a Bss HII site was created for the junction with khc, chosen because it minimally altered the Ncd neck according to the computer program COILS (6), changing L345A. The khc primers contained a Bss HII site, sequences coding for residues 338 to 340 missing from the khc plasmid, and an Eco RI site for ligation to pGEX-2T. pGEX/ncdkhc2 and -3 were made by replacing the Nsi I-Eco RI fragment of the plasmid with overlap extension PCR fragments [S. N. Ho, H. D. Hunt, R. M. Horton, J. K. Pullen, L. R. Pease, Gene 77, 51 (1989)] to substitute Ncd residues 664 to 700 for KHC residues 327 to 340 or to delete KHC after residue 326 and add a glycine. pGEX/ncdkhc1, -4, and -5 were made from pGEX/ncdkhc2 by replacing the Sph I-Nsi I fragment with overlap PCR fragments that repaired the L345A mutation in the Ncd neck or shifted the neck-motor junction by two residues or both. pGCX/ncdkhc6 was made from pGEX/MC1 (73) and wild-type khc by replacing the pGEX/MC1 Bst EII-Eco RI fragment with an overlap PCR fragment coding for Ncd to residue 663, followed by KHC residues 327 to 340. All plasmids were sequenced to confirm the intended changes and exclude PCR mutations.
19. The pGEX/ncdkhc1-5 chimeric plasmids were constructed from a plasmid coding for GST fused to Ncd residues 194 to 346 [S. A. Endow, S. Henikoff, L. Soler-Niedziela, Nature 345, 81 (1990)], followed by Drosophila KHC residues 11 to 340 [J. T. Yang, R. A. Laymon, L S. B. Goldstein, Cell 56, 879 (1989)]. The plasmid was constructed by using the polymerase chain reaction (PCR) and wild-type ncd and khc plasmids [H. Song and S. A. Endow, Biochemistry 35, 11203 (1996)]. A Bam HI site was created to ligate ncd to pGEX-2T [D. B. Smith and K. S. Johnson, Gene 67, 31 (1988)] and a Bss HII site was created for the junction with khc, chosen because it minimally altered the Ncd neck according to the computer program COILS (6), changing L345A. The khc primers contained a Bss HII site, sequences coding for residues 338 to 340 missing from the khc plasmid, and an Eco RI site for ligation to pGEX-2T. pGEX/ncdkhc2 and -3 were made by replacing the Nsi I-Eco RI fragment of the plasmid with overlap extension PCR fragments [S. N. Ho, H. D. Hunt, R. M. Horton, J. K. Pullen, L. R. Pease, Gene 77, 51 (1989)] to substitute Ncd residues 664 to 700 for KHC residues 327 to 340 or to delete KHC after residue 326 and add a glycine. pGEX/ncdkhc1, -4, and -5 were made from pGEX/ncdkhc2 by replacing the Sph I-Nsi I fragment with overlap PCR fragments that repaired the L345A mutation in the Ncd neck or shifted the neck-motor junction by two residues or both. pGCX/ncdkhc6 was made from pGEX/MC1 (73) and wild-type khc by replacing the pGEX/MC1 Bst EII-Eco RI fragment with an overlap PCR fragment coding for Ncd to residue 663, followed by KHC residues 327 to 340. All plasmids were sequenced to confirm the intended changes and exclude PCR mutations.
20. 550 of 0.65 to 1 and induced at 21° to 22°C. Induction times, usually 5 to 6 hours, were optimized by analysis on polyacrylamide gels. The molecular weight of the major induced protein band in each case corresponded to the predicted molecular weight of the hybrid protein. Lysates for motility assays were prepared as described in (15). Microtubule gliding assays were carried out by video-enhanced differential interference contrast (VE-DIC) microscopy [R. A. Walker et al., J. Cell Biol. 107, 1437 (1988)]. Samples of cell lysates used in motility experiments were analyzed on immunoblots. Proteins were reacted with antibody to HIPER, directed against a highly conserved sequence motif in the kinesin motor domain [K. E. Sawin, T. J. Mitchison, L. G. Wordeman, J. Cell Sci. 101, 303 (1992)] (gift of K. Sawin, ICRF, London) or an antibody to Ncd COOH-terminus residues 670 to 682 [M. Hatsumi and S. A. Endow, J. Cell Sci. 103, 1013 (1992)] and detected with an alkaline phosphatase system. A single major band of the predicted molecular weight was observed in each case, indicating that the chimeric proteins in the lysates used for the motility assays were intact.
21. 550 of 0.65 to 1 and induced at 21° to 22°C. Induction times, usually 5 to 6 hours, were optimized by analysis on polyacrylamide gels. The molecular weight of the major induced protein band in each case corresponded to the predicted molecular weight of the hybrid protein. Lysates for motility assays were prepared as described in (15). Microtubule gliding assays were carried out by video-enhanced differential interference contrast (VE-DIC) microscopy [R. A. Walker et al., J. Cell Biol. 107, 1437 (1988)]. Samples of cell lysates used in motility experiments were analyzed on immunoblots. Proteins were reacted with antibody to HIPER, directed against a highly conserved sequence motif in the kinesin motor domain [K. E. Sawin, T. J. Mitchison, L. G. Wordeman, J. Cell Sci. 101, 303 (1992)] (gift of K. Sawin, ICRF, London) or an antibody to Ncd COOH-terminus residues 670 to 682 [M. Hatsumi and S. A. Endow, J. Cell Sci. 103, 1013 (1992)] and detected with an alkaline phosphatase system. A single major band of the predicted molecular weight was observed in each case, indicating that the chimeric proteins in the lysates used for the motility assays were intact.
22. 550 of 0.65 to 1 and induced at 21° to 22°C. Induction times, usually 5 to 6 hours, were optimized by analysis on polyacrylamide gels. The molecular weight of the major induced protein band in each case corresponded to the predicted molecular weight of the hybrid protein. Lysates for motility assays were prepared as described in (15). Microtubule gliding assays were carried out by video-enhanced differential interference contrast (VE-DIC) microscopy [R. A. Walker et al., J. Cell Biol. 107, 1437 (1988)]. Samples of cell lysates used in motility experiments were analyzed on immunoblots. Proteins were reacted with antibody to HIPER, directed against a highly conserved sequence motif in the kinesin motor domain [K. E. Sawin, T. J. Mitchison, L. G. Wordeman, J. Cell Sci. 101, 303 (1992)] (gift of K. Sawin, ICRF, London) or an antibody to Ncd COOH-terminus residues 670 to 682 [M. Hatsumi and S. A. Endow, J. Cell Sci. 103, 1013 (1992)] and detected with an alkaline phosphatase system. A single major band of the predicted molecular weight was observed in each case, indicating that the chimeric proteins in the lysates used for the motility assays were intact.
23. 550 of 0.65 to 1 and induced at 21° to 22°C. Induction times, usually 5 to 6 hours, were optimized by analysis on polyacrylamide gels. The molecular weight of the major induced protein band in each case corresponded to the predicted molecular weight of the hybrid protein. Lysates for motility assays were prepared as described in (15). Microtubule gliding assays were carried out by video-enhanced differential interference contrast (VE-DIC) microscopy [R. A. Walker et al., J. Cell Biol. 107, 1437 (1988)]. Samples of cell lysates used in motility experiments were analyzed on immunoblots. Proteins were reacted with antibody to HIPER, directed against a highly conserved sequence motif in the kinesin motor domain [K. E. Sawin, T. J. Mitchison, L. G. Wordeman, J. Cell Sci. 101, 303 (1992)] (gift of K. Sawin, ICRF, London) or an antibody to Ncd COOH-terminus residues 670 to 682 [M. Hatsumi and S. A. Endow, J. Cell Sci. 103, 1013 (1992)] and detected with an alkaline phosphatase system. A single major band of the predicted molecular weight was observed in each case, indicating that the chimeric proteins in the lysates used for the motility assays were intact.
24. Axoneme-microtubule complexes were prepared as described in (15). To determine gliding velocities, we tracked axoneme-microtubule complexes with a custom tracking program (a gift of N. Gliksman and T. Salmon, University of North Carolina, Chapel Hill, NC).
25. R. Chandra, E. D. Salmon, H. P. Erickson, A. Lockhart, S. A. Endow, J. Biol. Chem. 268, 9005 (1993).
26. T. Q. P. Uyeda, P. D. Abramson, J. A. Spudich, Proc. Natl. Acad. Sci. U.S.A. 93, 4459 (1996).
27. H. Song, M. Golovkin, A. S. N. Reddy, S. A. Endow, ibid., 94, 322 (1997).
28. Supported by a grant from NIH. E. Sablin and R. Fletterick made valuable comments on constructs and motor structure. We thank H. Song for axonemes and for helpful comments on the manuscript.