A hydrophobic protein sequence can override a nuclear localization signal independently of protein context.

Abstract

Simian virus 40 T antigen is specifically targeted to the nucleus by the signal Pro-Lys-Lys-128-Lys-Arg-LysVal. We have previously described the isolation of a simian virus 40 T-antigen mutant, 676FS, which retains a wild-type nuclear localization signal but fails to accumulate properly in the nucleus and interferes with the nuclear localization of heterologous proteins. Here we report that the hydrophobic carboxy-terminal sequence novel to 676FS T antigen overrides the nuclear localization signal if fused to other proteins, thereby anchoring the proteins in the cytoplasm. We discuss possible mechanisms by which missorting of such a fusion protein could interfere with the nuclear transport of heterologous proteins. (4,20,27,34). While it was originally assumed that smaller nuclear proteins (<20 to 30 kDa) (14) would behave like small inert macromolecules and diffuse passively through the aqueous channel of the nuclear pore complex, a recent report by Breeuwar and Goldfarb (6) demonstrated that smaller nuclear proteins also use a receptor-facilitated import pathway. NLS-binding proteins have been detected by various assays in both nuclear and cytoplasmic compartments (1,33,54,59; for reviews, see references 17 and 52), and at least one cytoplasmic proteinaceous factor has been shown recently to be required for binding of nuclear proteins to the nuclear envelope in a cell-free assay (38)

Deciphering the Nuclear Import Pathway for the Cytoskeletal Red Cell Protein 4.1R

“…This peptide is necessary for nuclear import of 4.1R 80 , as shown by the predominantly cytoplasmic localization of 4.1R 80 mutKKK mutant and of 4.1G and 4.1N, two recently identified proteins sharing very high sequence homology with 4.1R but lacking a motif similar to the 4.1R NLS Walensky et al, unpublished data). However, this motif is not sufficient to target PK to the nucleus, consistent with other observations that the function of weak core NLS motifs requires other regions of the protein, either in proximity to the NLS or more distant from it (Rihs and Peters, 1989;Rihs et al, 1991;Hong and Engler, 1991;van Zee et al, 1991;Zhou et al, 1991;Gao and Knipe, 1992;Gashler et al, 1993;Jans and Jans, 1994;Schmolke et al, 1995;Douglas and Quinlan, 1996;Knuehl et al, 1996). Our study shows that this is also the case for protein 4.1R NLS, because the 30-kDa domain appears essential for optimal NLS function.…”

“…In the case of 4.1R 80 , however, the negatively charged EED motif is located much further upstream of the NLS. Precedence for influence of distant regions on function of an NLS motif has been reported previously (van Zee et al, 1991;Gao and Knipe, 1992). Second, we cannot rule out that the 30-kDa domain contains additional conformational determinants that contribute to the function of the downstream NLS.…”

“…However, the recent identification of two potent NLSs with novel sequences (Siomi and Dreyfuss, 1995;Pollard et al, 1996;Michael et al, 1997) and the characterization of numerous types and isoforms of shuttle proteins (Miyamoto et al, 1997;for review, see Gö rlich, 1998;Wozniak et al, 1998) have revealed additional complexity and diversity in nuclear import machinery. In some instances, other regions, either immediately flanking the NLSs or more distant from them, may override or activate the NLSs (Rihs and Peters, 1989;Rihs et al, 1991;Hong and Engler, 1991;van Zee et al, 1991;Zhou et al, 1991;Gao and Knipe, 1992;Gashler et al, 1993;Jans and Jans, 1994;Schmolke et al, 1995;Douglas and Quinlan, 1996;Knuehl et al, 1996).…”

Targeting of Proteins Derived from Self‐Processing Polyproteins Containing Multiple Signal Sequences

“…Introduction of a post‐translational NLS within the protein downstream of 2A (pPDF63; Table 1) did not, however, result in the cyan fluorescence localising to the nucleus, but retaining its mitochondrial pattern. ‘Over‐riding’ a NLS by the inclusion of a second targeting domain has been described previously (20–22).…”

“…Furthermore, by switching the order in which fluorescent proteins were encoded within the polyprotein (pPDF21), the same effect was observed (Table 1) Table 1) did not, however, result in the cyan fluorescence localising to the nucleus, but retaining its mitochondrial pattern. 'Over-riding' a NLS by the inclusion of a second targeting domain has been described previously (20)(21)(22).…”