Molecular segments of protein Tpr that confer nuclear targeting and association with the nuclear pore complex.
Tpr is a 267-kDa protein of unknown function recently identified as a constitutive component of the nuclear pore complex (NPC2)-attached intranuclear filaments. Secondary structure predictions suggest that the protein is divided into a large, coiled-coil forming aminoterminal domain and a shorter, highly acidic carboxyterminal domain. To identify which of Tpr's molecular segments determine its specific intranuclear localization, we have constructed expression vectors encoding various Tpr deletion mutants as well as chimeric combinations of Tpr sequences with the soluble cytoplasmic protein pyruvate kinase. Following transfection of cultured mammalian cells, we have identified a short region within Tpr's carboxyterminal domain that is essential and sufficient to mediate nuclear import of Tpr and which can also confer nuclear accumulation of pyruvate kinase. Tpr deletion mutants that contain this nuclear targeting segment, but lack the aminoterminal domain, appear evenly dispersed throughout the nucleus without any noticeable association to the NPC. In contrast, the aminoterminal domain lacking the carboxyterminal region remains located within the cytoplasm, forming aggregate-like structures not associated with the nuclear envelope. However, when tagged to Tpr's short nuclear targeting segment or to the nuclear localization signal of the SV40 large T protein, the aminoterminal domain is imported into the nucleus, where it then associates with the NPC. This association is mediated by shorter molecular segments within the aminoterminal domain which contain clusters of heptad repeats, whereas other regions are dispensable. This assignment of different topogenetic properties to distinct molecular segments of Tpr will now allow the design of future experiments to study the protein's structural properties further and determine its actual function.
Cordes
V C
VC
Medical Nobel Institute, Karolinska Institutet, Stockholm, S-17177, Sweden. volker.cordes@cmb.ki.se
Hase
M E
ME
Müller
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D011770:Pyruvate Kinase / Q000378:metabolism; D014982:Xenopus laevis
D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; C073597:TPR protein, human; D011770:Pyruvate Kinase