Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus

HEADLINES

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Molecular genetic approach to chloroplast protein targeting SMITH, T. A. and KOHORN, B. D. (1994) Mutations in a signal sequence for the thylakoid membrane identify multiple protein transport pathways and nuclear suppressors !. Cell BioL 126, 365-374 The relatively recent ability of researchers to apply sophisticated molecular genetic techniques to the unicellular green alga Chlamydomonas reinhardtii has led to the hope that this organism might prove valuable to plant cell biology in much the same way as yeasts have to the rest of cell biology. This paper represents a significant step in that direction, as it addresses protein targeting in Chlamydomonas with genetic techniques that have been invaluable in elucidating aspects of protein trafficking in yeast and bacteria. Smith and Kohom used site.directed mutagenesis to engineer the chloroplast-encoded gene for cytochrome f (which is essential for photosynthetic growth) to encode a transit peptide that can no longer direct ~he integration of this protein into the thylakold membrane. Interestingly, they found plelotropic effects of the altered cytochrome f on the targeting of a subset of other thylakoid.locallzed proteins. They then selected for suppressors that returned the algae to photosynthetic growth. The suppressors, which reflected further mutations in the signal peptlde as well as alterations in nucleus-encoded genes, not only resulted in correct localization of cytochrome f but also alleviated the pleiotropic effects on the other proteins. The nucleus.encoded proteins involved in suppression of the thylakoidmistargeting mutation in cytochrome f remain to be identified, and this may be the most challenging aspect of the endeavour. Nonetheless, the application of this methodology to a photo. synthetic organism seems likely to lead to the identification of polypeptides involved in plastid prote;n targeting. It should also fuel the efforts of plant biologists to continue the development of molecular techniques in this potentially important model organism.

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Splicing a signal SRINIVASAN, M., EDMAN, C. F. and SCHULMAN, H. (1994) Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus I. Cell Biol. 126, 839-852 Many intracellular signalling pathways are mediated by protein kinases,,which often modify several target substrates in different locations. MulUfunctional Ca2÷-calmodulin kinase (CaM kinase) is one such enzyme, and is involved in regulation of a wide variety of processes in both the nucleus and cytoplasm. CaM kinase exists as a multimer of six to twelve 50-60 kDa subunits and the holoenzyme can be >500 kDa in size. The subunits are encoded by four closely related genes - a, 13,7 and ~ - and additional isoforms are c~eneratedfrom each of these by alternative splicing, which varies the size 'nd composition of a variable region of the protein. This paper describes the c~taracterizaUon of one isoform, 8B-CaM, which is targeted to the nucleus. The variable domain of this isoform contains an alternatively spliced nuclear localization signal, which is sufficient to target cytosolic isoforms to the nucleus when it is inserted into their variable domains. CaM kinase therefore provides an example of alternative splicing of a localization signal being used to target protein isoforms differentially. Interestingly, coexpression of nuclear and cytoplasmic isoforms showed that the presence of an excess of one type could a~ter the intracellular distribution of the other; for example, an excess of the nuclear isoform could shift the cytoplasmic isoform to the nucleus. This provides evidence that the holoenzyme con¶ains different subunit isoforms and that their localization signals are able to compete to determine the overall localization of the enzyme. It therefore introduces a potentially powerful mechanism for regulation of CaM kinase function based on differential localization of the enzyme depending on the relative proportions of the subunits present.

RNA localizing RNA KLOC, M. and ETKIN, L. D. (1994) Delocalization of Vgl mRNA from the vegetal cortex in Xenopus oocytes after destruction of Xlsirt RNA Science 26.5, 1101-1103 LocaU;~:ationof maternally derived macromolecules in the oocyte is an important element of embryonic development in a number of species. Recently,the molecular mechanisms involved have begun to be elucidated and they appear to be both complex and varied. In Xenopus, mRNAs localized to the vegetal pole of the oocyte include Vgl and Xcat2, whose products are implicated in patterning of the early embryo. The vegetal cortex also contains a group of heterogeneous nontranslatable repeat transcripts called Xlsirts. To exal~e the potential role of Xlsirts in localization of Vgl and Xcat2 mRNA, Klnc and Et~in destroyed the localized Xlsirts RNA by injection of antisense oligodeoxynucleotides. This resulted in delocalization, but not degradation, of the Vgl mRNA, which became dispersed in the cytoplasm. In contrast, the localization of the Xcat2 mRNA was unaffected. Vgl RNA is thought to be localized at the vegetal cortex as a result of a microtubule.dependent t~'ansport step, followed by microfilament-dependent anchoring. Therefore, it seems that Xlsirts are involved in the microfilament-dependent anchoring of Vgl mRNA at the vegetal cortex, and that Xcat2 localization is achieved by a different mechanism. While it is possible that Vgl and Xlsirts interact directly, a more likely scenario is that Xlsirts RNA is a crucial component in organizing the microfilament network to which Vgl and probably other mRNAs are attached.

TRENDS IN CELLBIOLOGYVOL. 4 NOVEMBER1994

Thismonth's headlineswere contributed by PieroMorandini, IsabelleRe),, Debbie Sweetand StevenTheg. 387