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Signal recognition particle protein 19 is imported into the nucleus by importin 8 (RanBP8) and transportin

Kellie A. Dean^1,2,*, Oliver von Ahsen², Dirk Görlich² and Howard M. Fried^1,

¹ Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
² Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Federal Republic of Germany
^* Present address: Department of Genetics, Duke University Medical Center, Box 3657, Durham, NC 27710, USA

View larger version (44K): [in a new window]   Fig. 1. Endogenous SRP19 is localized to the nucleus and the nucleolus. (A,B) SRP19 in HeLa cells was detected using indirect immunofluorescence followed by confocal microscopy (see Materials and Methods). Antibodies against fibrillarin and TRAP (Görlich et al., 1990) were used as controls for nucleolar and rough endoplasmic reticulum subcellular staining. SRP19 was found to be prominent in the nucleoli and in the nucleoplasm, noticeable especially in the paraformaldehyde-fixed cells. Images were collected using the 63x oil objective of a Leica DM IRB/E microscope and analyzed with Leica TCS NT software. Bar, 10 µm. (C) A western blot showing that the anti-SRP19 antibody at 1:2000 dilution detected only SRP19 (*) in a total HeLa cell lysate.

View larger version (41K): [in a new window]   Fig. 2. Interaction of SRP19 with nuclear transport receptors. Recombinant SRP19 and rpL23a, fused to two tandem z-tags (IgG-binding domain of protein A), were immobilized on IgG-sepharose and tested for binding of transport receptors from a HeLa cell extract. Where indicated, 10 µM RanQ69L[GTP] was added. RanQ69L is a GTPase-deficient Ran mutant that remains GTP-bound in the presence of cytoplasmic Ran GTPase-activating protein (RanGAP1) (Bischoff et al., 1994). After extensive washing, bound proteins were eluted, precipitated, and analyzed by SDS-PAGE, followed by immunoblotting with specific antibodies (top) or by Coomassie blue staining (bottom). Load in the bound fractions corresponds to 15x the starting material.

View larger version (34K): [in a new window]   Fig. 3. Transport receptors bind directly to SRP19 in a RanGTP-regulated manner. A GST-SRP19 fusion protein was immobilized on glutathione-sepharose and incubated with purified nuclear transport receptors, with and without RanQ69L[GTP] as described in Materials and Methods. After washing, bound proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE. (A) Proteins that bound to GST-SRP19 in the presence (+) and absence (-) of RanQ69L[GTP]. Note that binding of all receptors to SRP19 was greatly reduced in the presence of RanQ69L[GTP]. (B) Purified receptors, in the same order as in A, loaded as reference samples. The asterisk indicates GST-SRP19.

View larger version (68K): [in a new window]   Fig. 4. SRP19 is imported into the nucleus by importin 8 and transportin. Recombinant human rpL23a or human SRP19 were purified, labeled with fluorophores and used as substrates (2 µM final concentration) for import into the nuclei of permeabilized HeLa cells. Import receptors were also used at 2 µM, except importin (imp) and importin 8 (imp 8), which were both at 4 µM. Reactions were stopped by fixation after 10 minutes for SRP19 and 25 minutes for rpL23a and analyzed by confocal microscopy. As previously reported, rpL23a could be imported by any of four transport receptors: importin ß (impß; C), transportin (trn; D), importin 5 (imp5; E), and importin 7 (imp7; F) (Jäkel and Görlich, 1998). SRP19 import was most efficient with importin 8 (imp8; G); however, transportin also mediated nuclear import of SRP19 (D). All images were collected and analyzed as described in Fig. 1. Bar, 10 µm.