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First published online March 23, 2005
doi: 10.1242/10.1242/jcs.01739

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Nuclear import of the histone acetyltransferase complex SAS-I in Saccharomyces cerevisiae

Sigrid Schaper^*, Jacqueline Franke, Sebastiaan H. Meijsing and Ann E. Ehrenhofer-Murray^*

Otto-Warburg-Laboratories, Max-Planck-Institute of Molecular Genetics, Ihnestraße 73, 14195 Berlin, Germany

View larger version (51K): [in a new window]   Fig. 1. Protein-protein interactions within the SAS-I complex. (A) Sas4p interacted with Sas2p and Sas5p in the two-hybrid system as shown by growth or no growth of the reporter strain in the absence of histidine. GBD, Gal4 DNA-binding domain (pAS-BC); GAD, Gal4 activation domain (pACTIIst); see Table 2 for plasmid details. (B) Coimmunoprecipitation analysis of Sas5p-HA3/myc6-Sas2p, Sas4p-myc9/Sas2p and Sas5p-HA3/myc6-Sas4p interactions. Bound proteins were analyzed on 12.5% SDS polyacrylamide gels, immunoblotted and probed with -myc or -Sas2 antibodies. 2, SAS2; 4, SAS4; 5, SAS5. (C) GST-pulldown assay to determine direct interactions within the SAS-I complex. Purified GST-Sas fusion proteins were immobilized on glutathione agarose beads and incubated with in vitro-translated radiolabeled SAS-I proteins (abbreviated 2, 4 and 5) or luciferase (L, negative control). An asterisk indicates degradation products of radiolabeled Sas4p. Washed beads were eluted with sample buffer, separated on 12.5% SDS polyacrylamide gels, and bands were visualized using a PhosphoImager.

View larger version (31K): [in a new window]   Fig. 2. Sas2p and Sas5p interacted with the importins Pse1p and Kap123p. (A) Domains in Pse1p. The blue bar indicates the importin ß homology domain containing an Armadillo repeat (ARM, red). The C-terminal fragments of Pse1p interacting with Sas5p in the two-hybrid assay are illustrated by black lines. (B) Coimmunoprecipitation analysis of Sas5p-myc9/Pse1p-HA3, Sas5p-myc9/Kap123p-HA3, myc6-Sas2p/Pse1p-HA3 and myc6-Sas2p/Kap123p-HA3 interactions. Bound proteins were separated on 12.5% SDS polyacrylamide gels, immunoblotted and probed with an -HA antibody. Pse1p-HA3 coimmunoprecipitated with Sas5p-myc9 or myc6-Sas2p (upper panels), and Kap123p-HA3 co-precipitated with Sas5p-myc9 or myc6-Sas2p (lower panels).

View larger version (50K): [in a new window]   Fig. 3. Nuclear accumulation of GFP-Sas2p and GFP-Sas5p was significantly decreased by kap123 or pse1-1. GFP-Sas2p (pAE1098), GFP-Sas4p (pAE1101) or GFP-Sas5p (pAE1106) were expressed in wild-type (WT) and kap mutant strains (see Tables 1 and 2), and the GFP tag was detected by fluorescence microscopy. The Hoechst staining visualizes the nucleus. Bars, 5 µm.

View larger version (45K): [in a new window]   Fig. 4. Nuclear localization of GFP-Sas2p and GFP-Sas5p was impaired in kap123, but not in kap108, kap119, kap122 and kap142 strains. GFP-Sas2p, GFP-Sas4p or GFP-Sas5p (see Table 2) were detected in wild-type (WT) and kap deletion strains (as indicated) by fluorescence microscopy. Bar, 5 µm.

View larger version (27K): [in a new window]   Fig. 5. Deletion analysis of Sas2p, Sas4p and Sas5p and their cellular localizations. (A) Predicted domains, motifs and nuclear localization signals (NLS) of Sas2p, Sas4p and Sas5p. The indicated domains/motifs were found by sequence analysis with the Prosite motif scan and the SMART domain search program. NLSs are depicted as green triangles; an asterisk indicates that the NLS could not be verified experimentally. In Sas2p, the histone acetyltransferase (HAT) domain is shown (red), preceded by a zinc finger motif (yellow). A cullin homology domain (orange) was found in Sas4p. Sas5p has sequence homology to the YEATS family of proteins (blue hexagon). (B) The N-terminal domains of Sas2p and Sas5p were necessary for their nuclear accumulation. Full-length or fragments of Sas2p, Sas4p and Sas5p (pAE1098-pAE1108, Table 2) were expressed as fusions to GFP in wild-type yeast cells and visualized by fluorescence microscopy. The start and end point of each fusion is indicated by their corresponding amino acids to the left of each image. Bar, 5 µm.

View larger version (14K): [in a new window]   Fig. 6. Size fractionation of wild-type and pse1-1 protein extracts prepared from cells (AEY2956 or AEY2957) grown at 24°C and expressing HA-Sas5p (pAE625). The elution profiles of HA-Sas5p were analyzed by gel electrophoresis on 12.5% SDS polyacrylamide gels and immunoblotting of the indicated fractions using an anti-HA antibody. The elution peaks of marker proteins are labeled above.

View larger version (22K): [in a new window]   Fig. 7. Alignment of the suggested NLSs of Sas2p and histones H3 and H4. Histone H1, Snf12p and the histone variant Htz1p were found by BLAST P queries using the NLSs of Sas2p, histones H3 and H4, respectively, and showed significant similarity to the consensus sequence.

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