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Proteomic analysis of plant microtubule-associated proteins
Cell Biology International
Cell Biology International 27 (2003) 181
www.elsevier.com/locate/jnlabr/ycbir
Short communication
Proteomic analysis of plant microtubule-associated proteins Jordi Chan, Guojie Mao, Clive Lloyd * Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK Accepted 15 October 2002
Until quite recently, few plant microtubule-associated proteins (MAPs) had been identified, but several have now been characterized. One strategy for identification of MAPs has been to use antibodies and degenerate PCR primers to look for plant proteins that share sequences with the better-studied proteins of animals and yeast. This has yielded data, for example, on microtubule motor proteins. Another approach has been to characterize mutants defective in processes found to be dependent on microtubules. This has produced fra2/ bot1—cognate to the microtubule-severing protein, katanin—and the microtubule organization mutant, MOR1—cognate to Xenopus XMAP215. A third approach has been the direct biochemical isolation of proteins by microtubule affinity. This has yielded the MAP65 family (Chan et al., 1996; Jiang and Sonobe, 1993), which forms cross-links between microtubules (Chan et al., 1999). One member of this family, NtMAP651a, has recently been cloned (Smertenko et al., 2000). Our approach has been to identify MAPs by direct functional assay in the belief that some plant MAPs may have limited homology with other eukaryotes or with proteins not yet identified as MAPs. We have adapted our methods developed for carrot cell suspensions, to Arabidopsis suspensions. Microtubule-binding proteins were trysinized and their peptide mass analysed by MALDI and compared in silico to the theoretical peptide mass fingerprints of proteins coded by the Arabidopsis genome. In this way, we have identified a range of proteins. In addition to proteins expected to co-purify with microtubules, such as Spc98p, MOR1, elongation factor 1alpha, MAP65 and several kinesins, we have identified several unknown proteins that are under study. In a related investigation, we have used the simple cell system reported inBarroso et al. (2000). By subculturing * Corresponding author. Tel. +44-1603-452-571; fax: +44-1603-501-771.
carrot suspension into medium without 2,4-D, the cells stop dividing and elongate. They no longer contain the mitotic kinesins DcKRP120-1 and -2, and express only cortical microtubules. This is an excellent system for investigating proteins required to make the interphase microtubule array. MAP65 isolated from dividing cell populations presents as three bands on 1-D SDS gels, but when isolated from elongated interphase cells shows only a single band. Analysis by mass spectrometry (Q-tof) reveals that this is a member of the most conserved subgroup, group1, of the multigene family, of which there are nine members in Arabidopsis. In an Arabidopsis cell suspension, the two MAP65 isoforms isolated on endogenous microtubules are also members of group1. These results demonstrate: the existence of an array-specific pattern of expression; that not all of the MAP65 family are required for the formation of parallel groups of microtubules in the interphase cortical array; and that the pattern of expression can be altered by changing the hormonal status.
References Barroso C, Chan J, Allan V, Doonan JH, Hussey PJ, Lloyd CW. Two kinesin-related proteins associated with the cold-stable cytoskeleton of carrot cells: characterization of a novel kinesin. Plant J 2000;24:859–68. Chan J, Rutten T, Lloyd CW. Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa MAP decorates all four microtubule arrays and the nucleus. Plant J 1996;10:251–9. Chan J., Jensen CG, Jensen LCW, Bush M, Lloyd CW. The 65-kDa carrot microtubule-associated protein forms regularly arranged filamentous cross-bridges between microtubules. Proc Natl Acad Sci USA 1999;96:14931–6. Jiang C-J, Sonobe S. Identification and preliminary characterization of a 65 kDa higher-plant microtubule-associated protein. J Cell Sci 1993;105:891–901. Smertenko A, Saleh N, Igarashi H, Mori H, Hauser-Hahn I, Jiang CJ et al. A new class of microtubule-associated proteins in plants. Nat Cell Biol 2000;2:750–3.
1065-6995/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1065-6995(02)00339-6
Cell Biology International 27 (2003) 181
www.elsevier.com/locate/jnlabr/ycbir
Short communication
Proteomic analysis of plant microtubule-associated proteins Jordi Chan, Guojie Mao, Clive Lloyd * Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK Accepted 15 October 2002
Until quite recently, few plant microtubule-associated proteins (MAPs) had been identified, but several have now been characterized. One strategy for identification of MAPs has been to use antibodies and degenerate PCR primers to look for plant proteins that share sequences with the better-studied proteins of animals and yeast. This has yielded data, for example, on microtubule motor proteins. Another approach has been to characterize mutants defective in processes found to be dependent on microtubules. This has produced fra2/ bot1—cognate to the microtubule-severing protein, katanin—and the microtubule organization mutant, MOR1—cognate to Xenopus XMAP215. A third approach has been the direct biochemical isolation of proteins by microtubule affinity. This has yielded the MAP65 family (Chan et al., 1996; Jiang and Sonobe, 1993), which forms cross-links between microtubules (Chan et al., 1999). One member of this family, NtMAP651a, has recently been cloned (Smertenko et al., 2000). Our approach has been to identify MAPs by direct functional assay in the belief that some plant MAPs may have limited homology with other eukaryotes or with proteins not yet identified as MAPs. We have adapted our methods developed for carrot cell suspensions, to Arabidopsis suspensions. Microtubule-binding proteins were trysinized and their peptide mass analysed by MALDI and compared in silico to the theoretical peptide mass fingerprints of proteins coded by the Arabidopsis genome. In this way, we have identified a range of proteins. In addition to proteins expected to co-purify with microtubules, such as Spc98p, MOR1, elongation factor 1alpha, MAP65 and several kinesins, we have identified several unknown proteins that are under study. In a related investigation, we have used the simple cell system reported inBarroso et al. (2000). By subculturing * Corresponding author. Tel. +44-1603-452-571; fax: +44-1603-501-771.
carrot suspension into medium without 2,4-D, the cells stop dividing and elongate. They no longer contain the mitotic kinesins DcKRP120-1 and -2, and express only cortical microtubules. This is an excellent system for investigating proteins required to make the interphase microtubule array. MAP65 isolated from dividing cell populations presents as three bands on 1-D SDS gels, but when isolated from elongated interphase cells shows only a single band. Analysis by mass spectrometry (Q-tof) reveals that this is a member of the most conserved subgroup, group1, of the multigene family, of which there are nine members in Arabidopsis. In an Arabidopsis cell suspension, the two MAP65 isoforms isolated on endogenous microtubules are also members of group1. These results demonstrate: the existence of an array-specific pattern of expression; that not all of the MAP65 family are required for the formation of parallel groups of microtubules in the interphase cortical array; and that the pattern of expression can be altered by changing the hormonal status.
References Barroso C, Chan J, Allan V, Doonan JH, Hussey PJ, Lloyd CW. Two kinesin-related proteins associated with the cold-stable cytoskeleton of carrot cells: characterization of a novel kinesin. Plant J 2000;24:859–68. Chan J, Rutten T, Lloyd CW. Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa MAP decorates all four microtubule arrays and the nucleus. Plant J 1996;10:251–9. Chan J., Jensen CG, Jensen LCW, Bush M, Lloyd CW. The 65-kDa carrot microtubule-associated protein forms regularly arranged filamentous cross-bridges between microtubules. Proc Natl Acad Sci USA 1999;96:14931–6. Jiang C-J, Sonobe S. Identification and preliminary characterization of a 65 kDa higher-plant microtubule-associated protein. J Cell Sci 1993;105:891–901. Smertenko A, Saleh N, Igarashi H, Mori H, Hauser-Hahn I, Jiang CJ et al. A new class of microtubule-associated proteins in plants. Nat Cell Biol 2000;2:750–3.
1065-6995/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1065-6995(02)00339-6