INTRODUCTION The nucleus of all eukaryotic cells from protozoa to mammals is delimited by a specialized double membrane, the nuclear envelope that is perforated by nuclear pore complexes (NPC). NPCs are the sites at which free diffusion of molecules smaller than 10 nm in diameter, and regulated trafficking of macromolecules in and out of the nucleus take place. In particular, transport of karyophilic proteins from their site of synthesis in the cytoplasm to their destination in the nucleus has been shown to occur via a number of signal-mediated pathways that share common features (Corbett and Silver, 1997; Nigg, 1997). Briefly, karyophilic proteins are endowed with a nuclear location signal (NLS) that is recognized by a receptor. The protein/receptor complex is actively transported through the NPC and, once in the nucleus, the complex dissociates and the receptor returns to the cytoplasm. There is heterogeneity both at the level of the NLS and of the specific receptors that recognize them. The prototypical NLS, exemplified by the SV40 large T antigen, is a seven amino acid peptide characterized by a high content of basic amino acids. A variation of this basic residue-rich NLS is the bipartite-type NLS originally identified in nucleoplasmin and that consists of two clusters of basic amino acids, with a high prevalence of lysines, separated by ten amino acids. A third type of NLS, termed M9, has been recently identified in the RNA binding protein hnRNPA1 and bears no similarity to the monopartite and bipartite NLS (Siomi and Dreyfuss, 1995). The heterogeneity of the NLS is reflected at the level of the transport receptors the different NLS interact with. A heterodimeric receptor consisting of importin α and importin β (also termed karyopherin α and β) binds to the classical NLS-bearing proteins and brings them to the transport apparatus located at the NPC. Importin α binds to the NLS, whereas importin β mediates binding to the NPC. In contrast, a single monomeric receptor, termed transportin, binds to M9 and mediates nuclear import of hnRNPA1 (Pollard et al., 1996). Importin β and transportin are distantly related in sequence and are members of a superfamily of proteins that are implicated as nuclear transport receptors. Other essential components involved in nuclear import of most karyophilic proteins are the small Ras-related GTP-ase, Ran/TC4, and its regulatory factors. There is so far only one example of Ran- independent transport, namely the HIV-1 Vpr protein, which seems to gain nuclear access by using a pathway distinct from the basic NLS and M9 pathway (Jenkins et al., 1998). In addition to the NPC architecture, many components of the nuclear transport apparatus have been conserved throughout 899 Journal of Cell Science 113, 899-906 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JCS1034 In all eukaryotic organisms proteins are targeted to the nucleus via a receptor-mediated mechanism that requires a specific nuclear localization sequence (NLS) in the protein. Little is known about this process in trypanosomatid protozoa that are considered amongst the earliest divergent eukaryotes. We have used the green fluorescent protein (gfp) and β-galactosidase reporters to identify the NLS of two trypanosomal proteins, namely the Trypanosoma brucei La protein homologue and histone H2B of T. cruzi. A monopartite NLS was demonstrated at the C terminus of the La protein, whereas a bipartite NLS was identified within the first 40 amino acids of histone H2B. Treatment of live trypanosomes with poisons of ATP synthesis resulted in exit of the La NLS-gfp fusion from the nucleus. Interestingly, this fusion protein accumulated at several discrete sites in the cytoplasm, rather than equilibrating between the nucleus and the cytoplasm. When ATP levels returned to normal, the protein reentered the nucleus, demonstrating that the process was energy dependent. Finally, using fusion proteins that localize to the nucleoplasm or the nucleolus, we identified a subpopulation of mitotic cells in which the chromosomes have segregated but the daughter nuclei remain connected by a thin thread-like structure. We propose that cells containing this structure represent a late stage in nuclear division that can be placed after chromosome segregation, but before completion of karyokinesis. Key words: Trypanosome, Nuclear protein, NLS, La, H2B SUMMARY Import of proteins into the trypanosome nucleus and their distribution at karyokinesis Maria A. Marchetti 1 , Christian Tschudi 1 , Helen Kwon 1 , Sandra L. Wolin 2,3 and Elisabetta Ullu 1,2, * Departments of 1 Internal Medicine and 2 Cell Biology, and 3 Howard Hughes Medical Institute, Yale University School of Medicine, PO Box 208022, 333 Cedar Street, New Haven, CT 06520-8022, USA *Author for correspondence (e-mail: elisabetta.ullu@yale.edu) Accepted 13 December 1999; published on WWW 14 February 2000