A Nuclear Export Signal Prevents Saccharomyces cerevisiae Hsp70 Ssb1p from Stimulating Nuclear Localization Signal-directed Nuclear Transport

Abstract

Hsp70 has been implicated in nuclear localization signal (NLS)-directed nuclear transport. Saccharomyces cerevisiae contains distinct SSA and SSB gene families of cytosolic Hsp70s. The nucleocytoplasmic localization of Ssa1p and Ssb1p was investigated using green fluorescent protein (GFP) fusions. Whereas GFP-Ssa1p localized both to the nucleus and cytoplasm, GFP-Ssb1p appeared only in the cytosol. The C-terminal domain of Ssb1p contains a leucine-rich nuclear export signal (NES) that is necessary and sufficient to direct nuclear export. The accumulation of GFP-Ssb1p in the nuclei of xpo1-1 cells suggests that Ssb1p shuttles across the nuclear envelope. Elevated levels of SSA1 but not SSB1 suppressed the NLS-GFP nuclear localization defects of nup188-⌬ cells. Studies with Ssa1p/Ssb1p chimeras revealed that the Ssb1p NES is sufficient and necessary to inhibit the function of Ssa-or Ssb-type Hsp70s in nuclear transport. Thus, NES-less Ssb1p stimulates nuclear transport in nup188-⌬ cells and NES-containing Ssa1p does not. We conclude that the differential function of Ssa1p and Ssb1p in nuclear transport is due to the NES-directed export of the Ssb1p and not to functional differences in their ATPase or peptide binding domains.The import of proteins into nuclei is mediated by soluble nuclear localization signal (NLS) 1 receptors. SV40 large Tantigen-like NLSs are bound in the cytoplasm by karyopherin ␣ (Kap ␣), which serves as an adapter to link NLS-cargo to karyopherin ␤ (Kap ␤). Kap ␤ mediates docking at the cytoplasmic face of the nuclear pore complex (1). Saccharomyces cerevisiae contains a single Kap ␣ gene encoded by SRP1. Translocation of the NLS-cargo-Kap ␣/␤ ternary complex occurs through the central channel of the nuclear pore complex. Once in the nucleus, the NLS-cargo dissociates and Kap ␣ and ␤ are recycled to the cytoplasm (1, 2). Only a portion of the cellular import traffic is mediated by Kap ␣/␤ heterodimers. Other classes of NLS-cargo, for example shuttling pre-mRNA binding proteins that display M9-type import signals (3), are transported by Kap ␤-like factors that bind directly to cognate NLS-cargo without the aid of Kap ␣ adapters (1, 4). Hsp70/Hsc70s (collectively referred to here as Hsp70s) are conserved molecular chaperones that participate in a variety of cellular functions, including protein folding and transport and the repair of stress-induced damage (5-7). Hsp70s are composed of a 44-kDa N-terminal ATPase domain, an 18-kDa peptide binding domain, and a C-terminal 10-kDa variable domain of unknown function (8, 9). S. cerevisiae contains two families of cytosolic Hsp70 genes, SSA1-4 and SSB1-2 (10, 11). The yeast Ssa-type Hsp70s are similar to the cytosolic Hsp70s found in other organisms including bacteria. To date, Ssb-type Hsp70s have been identified only in fungi. In S. cerevisiae, Ssb1 and Ssb2 are associated with translating ribosomes and can be cross-linked to nascent polypeptides (12, 13).Hsp70s have been proposed to function in NLS-directed nuclear transport by promoting the formati...

Nuclear Pore Complex Function in Saccharomyces cerevisiae Is Influenced by Glycosylation of the Transmembrane Nucleoporin Pom152p

“…Plates were incubated at 32.5°for 72 hr. nuclear import (Shulga et al 1996(Shulga et al , 1999. To ascertain whether the suppression of nup1D temperature sensitivity that we observed was the result of increased Ssa1p, we overexpressed SSA1 in wild-type and nup1D cells and assayed for growth at permissive and nonpermissive temperatures ( Figure 4C).…”

The Hsp70-Ydj1 Molecular Chaperone Represses the Activity of the Heme Activator Protein Hap1 in the Absence of Heme

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Divergent Functional Properties of the Ribosome-Associated Molecular Chaperone Ssb Compared with Other Hsp70s

“…In fact, previous results indicate a role for the C terminus of Ssb in cellular localization, as the C terminus of Ssb has a nuclear export signal (NES). Although nearly all wild-type Ssb is found in the cytoplasm, Ssb lacking either the NES or the entire C terminus is found distributed throughout both the nucleus and cytoplasm (Shulga et al, 1999). However, because Ssb lacking the C terminus is functional, active export of Ssb from the nucleus is not required.…”