RPL25GFP cells have a specific defect in cotranslational translocation. (A) Yeast strains RS453 (wild-type), sec61-3, RPL25GFP (MPY6), and Δrpl25::pRPL25 (MPY69) carrying the PHO8-URA3 reporter plasmid (pMP211) were plated in parallel on synthetic media lacking either tryptophan (-trp; selects for plasmid) or tryptophan and uracil (-trp -ura; selects for reporter) and grown for 2 d at 30°C. (B) Yeast strains W303α (wild-type), sec62-1, sec65-1, and RPL25GFP, possessing the CPY-URA3 reporter plasmid (pMR12), were plated on synthetic media lacking either leucine (-leu) or leucine and uracil (-leu -ura). (C) Wild-type (RS453), RPL25GFP, sec65-1 and sec61-3 transformed with pMP220 (PHO8-URA3-myc) were pulse-labeled for 5 min with [³⁵S]methionine at either 25 or 37°C and lysed, and then radiolabeled proteins were immunoprecipitated with α-myc antibodies and analyzed by SDS-PAGE and phosphorimaging. Positions of g-Pho8-Ura3p (membrane integrated) and Pho8p-Ura3p (cytosolic) are indicated. (D) Wild-type (RS453), RPL25GFP, and sec61-3 cells were pulse-labeled as in C and immunoprecipitated with α-CPY antibodies. Positions of g-pCPY (p1-luminal) and ppCPY (cytosolic) are indicated.

60S subunit biogenesis defects do not affect cotranslational translocation. (A) Extracts from MPY69 (Δrpl25[pRPL25]), RPL25GFP (MPY6) and Δrpl39 cells were centrifuged on 10–50% linear sucrose gradients and fractionated with continuous monitoring of A_254nm. Positions of 40S, 60S, 80S, and polysomes are indicated along with the presence of halfmers (*). (B) BY4742 (wild-type), Δrpl39, and RPL25GFP cells, carrying the PHO8-URA3 reporter plasmid (pMP234), were plated on synthetic media lacking either leucine (-leu) or leucine and uracil (-leu -ura).

Compromised NAC function does not lead to cotranslational targeting defects. (A) Yeast strains BY4742 (wild-type), Δegd1, Δegd2, Δbtt1, and RPL25-GFP, all transformed with the PHO8-URA3 reporter plasmid (pMP234), were streaked in parallel on synthetic media lacking either leucine (-leu; selects for plasmid) or leucine and uracil (-leu -ura; selects for reporter) and incubated for 2 and 4 d, respectively, at 30°C. (B) Yeast strains BY4742 (wild-type), Δegd1, Δegd2, Δbtt1, Δzuo1, and RPL25-GFP, all transformed with the PHO8-URA3-myc reporter plasmid (pMP220), were pulse labeled for 5 min with [³⁵S]methionine at either 30°C and lysed, and then radiolabeled proteins were immunoprecipitated with α-myc antibodies and analyzed by SDS-PAGE and phosphorimaging. Positions of g-Pho8-Ura3p (membrane integrated) and Pho8p-Ura3p (cytosolic) are indicated.

RPL25GFP cells have defects in SRP-ribosome binding. (A) Cell extracts from wild-type and RPL25GFP cells (1 OD₆₀₀) were solubilized with 1% CHAPS under high-salt (500 mM KOAc) conditions. After removal of unsolubilized material, ribosomes were isolated by centrifugation at 200 000 × g, and the total, pellet, and supernatant fractions were then analyzed by Western blot with Sec61p, Srp72p, and Rps3p antisera. (B) Ribosomal fractions from wild-type, RPL25GFP, and sec65-1 cells shifted to 37°C for 3 h, were prepared as in A; equivalent amounts of total extracts and ribosomal pellets were analyzed by Western blot with Sec61p and Rps3p antisera.

RPL25GFP cells show up-regulation of heat-shock chaperones. (A) Total cell extracts, prepared from wild-type (RS453), Δrpl39, and RPL25GFP cells were analyzed by SDS-PAGE and stained with Coomassie brilliant blue. Indicated bands (*) were excised and identified by mass spectrometry as follows: Hsp104p (accession no. P31539; 26 matching peptides), Sti1p (P15705; 18 peptides), Ssa1p (P10591; 28 peptides), Ssa2p (P10592; 31 peptides), and Hsp60p (P19882; 21 peptides). (B) Extracts from wild-type (RS453) and RPL25GFP cells were analyzed by Western blot with Ssa1p, Zwf1p, and Rps3p antibodies. (C) Fivefold serial dilutions of RPL25-GFP, Δzuo1 strains and isogenic wild-type strains (RS453 and BY4742, respectively) were spotted on to YPD or YPD supplemented with 1 mg · ml⁻¹ AZC and incubated for 2 and 3 d, respectively, at 30°C.

SRP overexpression partially rescues RPL25GFP phenotypes. (A) Wild-type (RS453) and RPL25GFP cells transformed with pMW295 and pMW299 (+SRP↑) or pRS425 and pRS426, were grown to early log phase in minimal media lacking leucine and uracil (-leu -ura) and diluted to an OD₆₀₀ of 0.1, and then fivefold serial dilutions were spotted on -leu -ura plates and grown for 3 d at 30°C. (B) The same strains as in A were grown at 30°C in liquid minimal media lacking leucine and uracil to midlog phase, diluted in the same media to an OD600 of 0.1, and then growth monitored over time. (C) Ribosome pellet fractions (prepared as in Figure 4A) from wild-type (RS435) and RPL25GFP cells, both transformed with pRS425 and pRS426, as well as RPL25GFP cells transformed with pMW295 and pMW299 (+SRP↑), were analyzed by Western blot with Srp72p and Rps3p antisera. (D) The same strains as in C were pulse-labeled with [³⁵S]methionine (as in Figure 1C) and immunoprecipitated with anti-DPAP B antibodies. g-DPAP B, glycosylated DPAP B; DPAP B, nonglycosylated DPAP B.