Identification of Two HSPT0-related Xenopus Oocyte Proteins That Are Capable of Recycling Across the Nuclear Envelope

Two 70-kD polypeptides, B3 and B4, are present in equivalent concentrations in the nucleus and cytoplasm of Xenopus oocytes. The objectives of this study were to determine if they (a) are members of the 70-kD family of heat shock proteins, and (b) recycle between the nuclear and cytoplasmic compartments. Evidence based on high-affinity binding to ATP, cross-reactivity of B3/B4-specific antibodies with rat hsc70, and a comparison of cyanogen bromide cleavage peptide maps with hsc70, verified that B3 and B4 are members of the 70-kD family of heat-shock proteins. Nuclear uptake studies were performed by microinjecting ~5I-labeled B3/B4, rat hsc70, and BSA into the cytoplasm of oocytes, and examining their subsequent intracellular distributions. By 6 h postinjection, the nuclear concentration of B3/B4 and hsc70 were ~24-fold greater than BSA controls. It was also found that B3/B4-coated gold particles as large as 120/~ in diameter were able to enter the nucleus by passing through the pores. Nuclear efflux was analyzed by microinjecting the iodinated proteins directly into the oocyte nuclei. 2 h after nuclear injection, at least 46% of the B3/B4 and 60% of the hsc70 were found in the cytoplasmic fractions, compared with <10% for the BSA controls. Cell fusion experiments, in which labeled, anucleate oocyte vegetal hemispheres were fused, under oil, with nucleate unlabeled animal hemispheres, demonstrated that cytoplasmic B3 and B4 could enter the nucleus after equilibration was reached, arguing against the existence of separate nuclear and cytoplasmic populations. Collectively, these results show that B3, B4, and rat hsc70 are transported across the nuclear envelope and recycle between the nucleus and cytoplasm. T wo prominent 70-kD Xenopus laevis oocyte proteins, designated B3 and B4 (pI 5.58 and 5.75, respectively), are present in equivalent concentrations in both the nucleus and cytoplasm (DeRobertis et al., 1978; Feldherr and Ogburn, 1980). These polypeptides are also present in adult Xenopus tissues, including liver, kidney, pancreas, brain, and heart (Dreyer and Hausen, 1983). Since B3 and B4 are not restricted to oocytes, it is likely that they have a universal role in cell function. King and Davis (1987) and Horrell et al. (1987) suggested that B3 and B4 might be constitutively expressed heat shock proteins, similar to the heat shock cognates (hsc70s) characterized in Drosophila (Craig et al., 1983). These conclusions were based on similarities in the molecular weights and isoelectric points of these proteins. Metabolically labeled B3 and B4 equilibrate between the nucleus and cytoplasm in •3 h (Feldherr and Ogburn, 1980). Exogenous proteins of equivalent size that are known to enter the nucleus by passive diffusion require a much longer time to equilibrate (e.g., BSA, Bonner, 1975; Paine, 1975; Paine et al., 1975), indicating that the uptake of B3 and B4 is transport mediated. There are two likely explanations why these polypeptides do not accumulate in the nucleoplasm like other targeted oocyte nuclear proteins, such as nucleoplasmin (Dingwall et al., 1988) and N1/N2 (Kleinschmidt and Seiter, 1988). First, only a portion of the cytoplasmic pool of B3 and B4 might be available to the nucleus. This could be the result of either binding or compartmentalization within the cytoplasm. Second, B3 and B4 might shuttle between the nucleus and cytoplasm. The objectives in this study were to determine whether B3 and B4 are constitutively expressed heat shock proteins, and if they are capable of recycling between the nucleus and cytoplasm. Both hsp70 and hsc70 are concentrated in the nucleus in response to heat or chemical stress (Welch and Feramisco, 1984; Pelham, 1984; Pelham, 1986) and apparently are redistributed back to the cytoplasm during recovery (Velaquez and Lindquist, 1984; Welch and Mizzen, 1988). It was not determined, however, whether these proteins are able to continuously recycle under nonstress conditions. Demonstrating that B3 and B4 are recycling heat shock cognate proteins could give useful clues as to the functions of this family of polypeptides, especially since they are so highly conserved (Lindquist and Craig, 1988). For example, recycling would be consistent with the hypothesis that these proteins act as carriers in signal-mediated nuclear transport. By analogy, hsp70 has been shown to be involved in the translocation of proteins across mitochondrial and endoplasmic reticulum membranes (Chirico et al., 1988; Deshaies et al., 1988). © The Rockefeller University Press, 0021-9525/90/11/1775/9 $2.00 The Journal of Cell Biology, Volume 111, November 199