Snl1 interacts with ribosomal proteins. (A) Amino- or carboxyl-terminally FLAG (F)-tagged NEFs were immunoprecipitated from wild-type (BY4741) whole-cell lysates as described in Materials and Methods. Immunoblot analysis was used to detect proteins interacting with anti-Ssa, anti-Ssb, and anti-Rpl8 antisera. Red letters represent proteins of the large (L) and small (S) subunits of the ribosome that were identified by using mass spectrometry. EV, empty vector. (B) The whole-cell lysate from BY4741 expressing Snl1ΔN-FLAG was split equally and incubated on ice for 15 min with the indicated concentrations of KCl. Snl1ΔN was immunoprecipitated, and interacting proteins were separated on a 15% SDS-PAGE gel. Immunoblot analysis was used to detect Rpl3 and Snl1ΔN (anti-FLAG antibody). CBB, Coomassie brilliant blue; IP, FLAG immunoprecipitation; IB, immunoblot of the whole-cell lysate.

Snl1 interacts with the ribosome via a lysine-rich-region binding motif. (A) Truncations within putative helix 1 of the Snl1 Bag domain are depicted. TM, putative transmembrane domain. The lysine-rich region between residues 50 and 60 in Snl1 is highlighted, and the residue changes that result in the 5KA mutant are indicated. (B) FLAG (F)-tagged Snl1ΔN truncations were immunoprecipitated from the wild-type (BY4741) whole-cell lysate. Immunoblot analysis was used to detect ribosomal proteins with anti-Rpl3 antisera. (C) FLAG-tagged Snl1ΔN, either the wild type (WT) or the 5KA mutant, was immunoprecipitated from BY4741 cells, and immunoblot analysis was used to detect stably produced proteins with anti-Rpl3 antisera. IP, FLAG immunoprecipitation; IB, immunoblot of the whole-cell lysate.

Snl1 binds to the 60S ribosome subunit independently of Hsp70. (A) Whole-cell lysates from the ssb1Δ ssb2Δ strain (Δ) or its isogenic wild-type (WT) (DS10) strain expressing either the empty vector (EV) or Snl1ΔN-FLAG were subjected to FLAG immunoprecipitation (IP). Immunoblot analysis was used to detect stably produced protein with anti-Ssb, anti-Rpl3, and anti-FLAG antisera. (B) FLAG-tagged Snl1ΔN, either the wild type or the E112A,R141A mutant, was immunoprecipitated from BY4741 cells. Immunoblot analysis was used to detect stably produced protein with anti-Ssa, anti-Ssb, anti-Rpl3, and anti-FLAG antisera. IB, immunoblot of the whole-cell lysate. (C) Snl1ΔN-FLAG expressed in BY4741 cells was immobilized onto M2 FLAG resin and incubated with His₆-Ssb (1 mg/ml) for 30 min on ice, followed by another round of immunoprecipitation. Bound proteins were eluted as described in Materials and Methods, and immunoblot analysis was performed to detect protein with anti-Rpl3 and anti-FLAG antisera. The Ssb antibody detected endogenous (bottom band) and recombinant (bottom band) Ssb present in the fractions. (D) Snl1ΔN-FLAG expressed in an Rps6B-3×HA strain was immobilized on M2 FLAG resin. The resin was equally split and treated with and without 40 mM EDTA for 30 min on ice to dissociate intact ribosomes. Bound proteins were eluted as described in the text, and immunoblot analysis was performed to detect protein with anti-HA, anti-Rpl8, and anti-FLAG antisera.

Ribosome association is conserved in the sole BAG domain-containing protein of Candida albicans. (A) Illustration depicting the sequence alignment of BAG domain-containing proteins of Saccharomyces cerevisiae and Candida albicans showing the lysine-rich region at the N terminus of putative helix 1 and the predicted transmembrane (TM) domains. Murine Bag-1 lacks both a transmembrane domain and a Lys-rich region. Mm, Mus musculus. (B) Localization of the chromosomally tagged C. albicans Snl1 homolog (CaSnl1-GFP) in wild-type C. albicans strain SC5314 was determined by fluorescence microscopy. Cells were stained with Hoechst 33342 dye, as described in Materials and Methods, to facilitate the assessment of perinuclear ER localization. (C) Snl1ΔN-FLAG from S. cerevisiae and the corresponding truncations in the C. albicans Snl1 homolog and the mammalian Bag-1 protein were expressed and immunoprecipitated from the wild-type (BY4741) yeast whole-cell lysate. Proteins were separated on a 15% SDS-PAGE gel, and immunoblot analysis was used to detect ribosomal proteins with anti-Rpl8 antisera. CBB, Coomassie brilliant blue; IP, FLAG immunoprecipitation; IB, immunoblot of the whole-cell lysate.

Hsp70 recruitment by ribosome-associated cofactors. Hsp70 protein chaperones are shown to be recruited to intact, translating ribosomes via interactions with ribosome-associated factors. Mpp11 and RAC (Zuo1) are J proteins, whereas Snl1 is a nucleotide exchange factor. Both proteins bind Hsp70 within the NBD and accelerate its intrinsic ATPase activity.