Differential importin-alpha recognition and nuclear transport by nuclear localization signals within the high-mobility-group DNA binding domains of lymphoid enhancer factor 1 and T-cell factor 1.

The transcription factor lymphoid enhancer factor 1 (LEF-1) is directed to the nucleus by a nine-amino-acid nuclear localization signal (NLS; KKKKRKREK) located in the high-mobility-group DNA binding domain. This NLS is recognized by two armadillo repeat proteins (pendulin/Rch1/alpha-P1/hSrp1alpha and Srp1/karyopherin-alpha/alpha-S1/NPI-1) which function in nuclear transport as the importin-alpha subunit of NLS receptors. T-cell factor 1 (TCF-1), a related transcription factor, contains a similar sequence (KKKRRSREK) in the identical position within its HMG DNA binding domain. We show that this sequence functions as an NLS in vivo but is not recognized by these two importin-alpha subtypes in a yeast two-hybrid assay and only weakly recognized in an in vitro binding assay. Transfer of the LEF-1 NLS to TCF-1 can confer pendulin/Rch1 binding, demonstrating that the NLS is the primary determinant for recognition. We have constructed a set of deletion mutations in pendulin/Rch1 to examine the differential NLS recognition more closely. We find that the entire armadillo repeat array of pendulin/Rch1 is necessary to maintain high affinity and specificity for the LEF-1 NLS versus the TCF-1 NLS. Importin-beta, the second subunit of the NLS receptor complex, does not influence in vitro NLS binding affinity or specificity. To test whether this differential recognition is indicative of distinct mechanisms of nuclear transport, the subcellular localization of LEF-1 and TCF-1 fused to green fluorescent protein (GFP)) was examined in an in vitro nuclear transport assay. GFP-LEF-1 readily localizes to the nucleus, whereas GFP-TCF-1 remains in the cytoplasm. Thus, LEF-1 and TCF-1 differ in several aspects of nuclear localization.

The nuclear localization signal of lymphoid enhancer factor-1 is recognized by two differentially expressed Srp1-nuclear localization sequence receptor proteins.

Proteins are directed to the nucleus by their nuclear localization sequences (NLSs) in a multistep process. The first step, which is to dock the NLS-containing protein to the nuclear pore, is carried out in part by a recently identified NLS receptor named Srp1/importin-alpha. Using the high mobility group (HMG) DNA binding domain of human lymphoid enhancer factor-1 (hLEF-1) as bait in a yeast two-hybrid screen, we have identified two different mouse Srp1 proteins (pendulin/importin-alpha and mSrp1) that each bind to a 9-amino acid sequence in hLEF-1 called the B box. We show that the B box of hLEF-1, a region essential for high affinity DNA binding, is also necessary and sufficient for nuclear localization, lending support to the model that NLSs can function both in nuclear transport and DNA binding. Pendulin and mSrp1 are the mouse homologues of hRch1/hSrp1alpha/importin-alpha and hSrp1/karyopherin alpha/NPI-1, respectively, and show considerable sequence divergence from each other. We find a surprising and significant difference in the expression pattern of pendulin and mSrp1 mRNA, suggesting that these two Srp1 proteins are distinguishable in function as well as sequence.

Xenopus interspersed RNA families, Ocr and XR, bind DNA-binding proteins.

Interspersed RNA makes up two-thirds of cytoplasmic polyadenylated RNA in Xenopus and sea urchin eggs. Although it has no known function, previous work has suggested that at least one family of interspersed RNA, XR, binds Xenopus oocyte proteins, and can influence the rate of translation. We have used two Xenopus repeat families, Ocr and XR, to explore their protein binding abilities. Ocr RNA binds the same pattern of highly abundant oocyte proteins that XR RNA binds, which are believed to be messenger ribonucleoprotein (mRNP) particle proteins. In addition, we show that Ocr RNA binds the Oct-60 protein, a member of the POU-domain family of transcription factors found in Xenopus oocytes. Using a 32 base pair sequence from the XR repeat in a DNA affinity column two proteins were isolated, 66 kDa and 92 kDa, that together form a complex with XR DNA. One of these proteins (92 kDa) also binds XR RNA. We suggest that the role of at least a subset of interspersed RNAs in development may be to bind, and sequester in the cytoplasm, DNA-binding proteins until the end of oogenesis.

Xenopus Gq alpha subunit activates the phosphatidylinositol pathway in Xenopus oocytes but does not consistently induce oocyte maturation.

We cloned the Xenopus laevis form of Gq alpha subunit to study its effects on oocyte maturation. Injection of Xenopus Gq alpha mRNA into stage 6 oocytes activated the phospholipase C/phosphatidylinositol pathway. The oocyte membrane became permeable to calcium ions and was able to generate transient inward currents (T(in)), due to the opening of Ca(2+)-dependent Cl- channels. The T(in) amplitude developed over several hours and disappeared by 24 hr. Diacylglycerol levels were found to parallel the appearance and disappearance of the T(in). The concurrent decline of T(in) values and diacylglycerol was not due to a failure in the synthesis of Gq alpha protein, which was produced continuously for > 24 hr. After Xenopus Gq alpha mRNA injection, germinal vesicle breakdown (GVBD) was variable (0-100%) in stage 6 oocytes, whereas none of the stage 4 oocytes underwent GVBD. In contrast, stage 6 oocytes injected with mRNA encoding the Go alpha G protein consistently underwent GVBD but did not acquire T(in). Our results show that activation of phospholipase C is not an absolute requisite for the induction of maturation, although in oocytes of some frogs phospholipase C activation can trigger a pathway to GVBD.