A plant in vitro system for the nuclear import of proteins

Abstract

This paper reports the development of an in vitro system that allows the direct assay of protein import into plant nuclei. In this assay the import of fluorescently labelled karyophilic protein substrates into nuclei isolated from evacuolated tobacco BY-2 suspension cells is monitored. It is demonstrated that import of the fluorescently labelled peptide conjugates is rapid, saturable and nuclear localization signal (NLS)-dependent. Exclusion of high molecular weight (70 kDa) dextran and substrates carrying mutated NLS sequences further underline the specificity of this system. Nuclear translocation of karyophilic import substrates in tobacco, similar to mammalian systems, is inhibited by the non-hydrolysable GTP analogue GTP-gamma-S. In contrast, protein uptake is not blocked by wheat germ agglutinin, N-ethyl-maleinimide and iodoacetic acid. Furthermore, it is shown that nuclear import of proteins is only partially inhibited by low temperature (0-4 degrees C). The in vitro nuclear import assay does not depend on exogenously added ATP or cytosolic factors. However, a block of nuclear import with GTP-gamma-S could be overcome by the addition of cytosolic extract, suggesting the dependence on cytosolic factors or proteins. These data indicate that the characteristics of nuclear protein import in plant and mammalian cells are similar, but may be, at least in some respects, also different from each other.

Protein Targeting to the Nuclear Pore. What Can We Learn from Plants?1

“…Recently, an in vitro import system that directly measures NLS protein import was developed and characterized using evacuolated permeabilized protoplasts derived from tobacco suspension cultures (Hicks et al, 1996;Merkle et al, 1996). Typically, vacuoles are rather fragile organelles containing high levels of hydrolytic activity and account for 80% of the total cell volume.…”

“…We used evacuolated protoplasts in these experiments because they are very stable, fully viable, and easy to work with. Permeabilization in these experiments was achieved by lowering the osmoticum in the medium (Hicks et al, 1996) or by adding low amounts of Triton X-100 (Merkle et al, 1996). In this system, fluorescently labeled NLS substrates were added to the permeabilized protoplasts and import was measured by fluorescence microscopy.…”

“…In addition, immunofluorescence and biochemical studies demonstrate that importin ␣ is tightly associated with the nucleus and probably the NPC (Smith et al, 1997). Indirect evidence also suggests that Ran is not fully depleted from this system (Merkle et al, 1996). Thus, it is possible that all of the NLS import factors remain associated with the nucleus, and possibly other structures in the cytoplasm, after the protoplasts are permeabilized.…”

“…Another unique feature of the plant import process is that NLS protein import can occur at 4°C (Hicks et al, 1996;Merkle et al, 1996). It is interesting that import into chloroplasts (Leheny and Theg, 1994) and mitochondria (Knorpp et al, 1994) in higher plants also occurs at 4°C, indicating that plants evolved mechanisms that allow these different import processes to occur at low temperatures.…”

“…The energy requirement for import in eukaryotes is probably conserved, because nuclear import can be blocked only by nonhydrolyzable GTP analogs in vertebrates and plants (Hicks et al, 1996;Merkle et al, 1996;Zupan et al, 1996;Gö rlich, 1997). Ran is necessary for GTP hydrolysis in vertebrates (Gö rlich, 1997), which strongly indicates a role for Ran in nuclear translocation that is functionally conserved in eukaryotes.…”