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. 2015 Jul 20;210(2):287-301.
doi: 10.1083/jcb.201410086.

Analysis of the interplay of protein biogenesis factors at the ribosome exit site reveals new role for NAC

Yvonne Nyathi  1 , Martin R Pool  2
Affiliations

Analysis of the interplay of protein biogenesis factors at the ribosome exit site reveals new role for NAC

Yvonne Nyathi et al. J Cell Biol. .

Abstract

The ribosome exit site is a focal point for the interaction of protein-biogenesis factors that guide the fate of nascent polypeptides. These factors include chaperones such as NAC, N-terminal-modifying enzymes like Methionine aminopeptidase (MetAP), and the signal recognition particle (SRP), which targets secretory and membrane proteins to the ER. These factors potentially compete with one another in the short time-window when the nascent chain first emerges at the exit site, suggesting a need for regulation. Here, we show that MetAP contacts the ribosome at the universal adaptor site where it is adjacent to the α subunit of NAC. SRP is also known to contact the ribosome at this site. In the absence of NAC, MetAP and SRP antagonize each other, indicating a novel role for NAC in regulating the access of MetAP and SRP to the ribosome. NAC also functions in SRP-dependent targeting and helps to protect substrates from aggregation before translocation.

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Figures

Figure 1.
Figure 1.
Association of Map1 with the ribosome at the UAS. (A) Extracts from WT cells (W303; T), were centrifuged through sucrose cushions at indicated salt concentrations to generate a ribosome-enriched pellet (P) and supernatant fraction (S). Fractions were analyzed by SDS-PAGE and blotting for Map1, Rpl17 (ribosome marker), and Zwf1 (cytosolic marker). (B and C) Ribosome pelleting analysis was performed as in A in the presence of 100 mM (B) or indicated salt concentrations (C) with prt1-1 (YMK135) cells grown either at 24°C or after a 20-min shift to 37°C followed by blotting for indicated proteins. (D) Ribosome pelleting was performed from WT (RS453), rpl25GFP and Δmap1 (GFY9) strains as in A. Pellets were analyzed by Western blot for Rpl25, Map1, and Zwf1 (loading control). (E) MetAP activity in total lysates from the indicated strains was determined using the fluorogenic substrate Met-AMC, as described in Materials and methods (error bars = SEM; n = 3). (F) Ribosome pellets from WT or a Δmap1::MAP1FLAG strain (GFY9::pMP289) were cross-linked with 250 µM MBS, followed by denaturing immunoprecipitation with anti-FLAG or control (α-myc) antibodies. Total reactions and immunoprecipitates (IP) were analyzed by Western blot for FLAG or Rpl35. The Map1xRpl35 cross-link is indicated (o).
Figure 2.
Figure 2.
Map1 and Map2 have distinct interactions with the ribosome. (A) WT and Δrpl25 cells complemented with either a plasmid-borne RPL25 (MPY69) or rpl25GFP (rpl25GFP) were spotted as serial dilutions onto YPD plates with or without 3 µM fumagillin and grown at 30°C for 2 d. (B) Ribosome pellets were prepared from WT (W303), and strains with C-terminally HA-tagged genomic MAP2 (MAP2HA) W303 (YNY5), rpl25GFP (YNY3), and Δmap1 (YNY4) and analyzed by Western blot for HA, Map1 and Rpl35. (C) Ribosome pellets from MAP2HA (YNY5) strain with or without Map1 overexpression plasmid (pTDH-MAP1, pMP299) were analyzed by Western blot for Map2HA, Map1, and Rpl25. The ribosome pellets from WT with (+) and without (−) the pTDH-MAP1 plasmid were analyzed by SDS-PAGE and Coomassie staining. The position of the Map1 protein is indicated. (D) Ribosome pellets prepared and analyzed as in B from either WT (W303) or MAP2HA (YNY5) strain, transformed with pRS414 (vector) or pMP305 (pGPD-MAP2HA). (E) Ribosome pelleting from MAP2HA (YNY5) cell extract was performed in the presence of either 100 mM or 500 mM salt. Resulting total (T), supernatant (S), and pellet (P) fractions were analyzed by blotting for Rpl35, Map2HA, and Map1.
Figure 3.
Figure 3.
Map1 is adjacent to NAC at the UAS. (A) Ribosome fractions derived from WT (BY4741) and Δnac strains were treated with the cross-linker GMBS (200 µM) and analyzed by Western blot for Map1. (B) Ribosome fractions from WT yeast (W303) with or without the pTDH-MAP1 plasmid (pMP299), as well a Δmap1 (GFY9) strain complemented with a Map1FLAG plasmid (pMP289), were treated with GMBS as in A and analyzed by Western blot for Egd2. (C) Cross-linking of ribosome fractions from WT (RPL35, W303) and a Δrpl35A/B strain expressing a C-terminally HA-tagged RPL35A (RBY175) were performed with DSS followed by Western blot for Egd2. (D) Ribosome pellets from indicated strains were analyzed by Western blot for Egd2, Rps3, and Rpl35.
Figure 4.
Figure 4.
Interplay of ribosome binding of SRP, NAC, and Map1. (A) Ribosome pellets, supernatant, and total cell extracts from WT cells, or WT cells transformed with plasmids to overexpress NAC (pMP304), Map1 (pMP299), or SRP (pMW295 and pMW299) were analyzed by Western blot for Sec65, Egd2, Map1, and Rpl35. (B) Serial dilutions of WT strain and the indicated nac mutants transformed with SRP overexpression were spotted on −URA/LEU plates and grown at 30°C for 3 d. (C) Ribosomes from WT cells (BY4741), Δegd1 and Δegd2 transformed with plasmids overexpressing SRP were analyzed by Western blot for Map1 and Rpl35. (D) WT (BY4741), Δegd1 and Δegd2 strains transformed with SRP overexpression plasmids or empty vectors and Δssb1/ssb2Δnac (control) were grown in minimal medium to mid-log phase. Lysates were extracted with nonionic detergent to yield a pellet enriched in detergent-insoluble aggregates, which were analyzed by SDS-PAGE and Coomassie staining. The amount of aggregates were quantified (error bars = SEM; n = 3; **, P < 0.01; ***, P < 0.001 one-way ANOVA). (E) Ribosome pellets from WT (BY4741), Δbtt1, Δegd1, and Δegd2 cells were analyzed by SDS-PAGE and blotted for Sec65, Egd1, Egd2, and Rpl35.
Figure 5.
Figure 5.
NAC mutants have mild SRP-dependent translocation defects. (A) WT (BY4741), Δegd1, Δegd2, Δbtt1, and rpl25GFP strains (control) were transformed with a PHO8-URA3 reporter plasmid (pMP234) and streaked onto SD–leu (selects for plasmid) or SD–leu –ura media (selects for translocation defect) and grown for 7 d at 16°C. (B) Translocation assay was performed as in A, but with CPY-URA3. (C) WT (W303), sec65-1 (CSY128), Δegd2, and sec65-1Δegd2 (YNY1) strains were grown to mid-log phase and serial dilutions spotted onto YPD media and grown at 30°C for 3 d. (D) PHO8-URA3 reporter assay was repeated as in A, but with strains as in C. (E) Total and ribosome fractions from cycloheximide-treated cells for strains as in C were analyzed by Western blot for Sec65, Rpl17, Egd1, and Egd2. (F) Dap2 translocation in WT (W303), sec65-1 (CSY128) cells, and sec65-1 cells overexpressing NAC (pMP304) or Egd2 (pMP302) was analyzed by pulse labeling. Nontranslocated (Dap2) and glycosylated (translocated) forms (g-Dap2) are indicated. (G) Ribosome pellets, prepared from WT and sec65-1 strains overexpressing Egd1, Egd2, or NAC were treated with DSS and analyzed by blotting for Egd2.
Figure 6.
Figure 6.
Egd2 prevents aggregation of nontranslocated SRP-dependent precursors. (A) WT (W303), Δegd1, Δegd2, sec65-1 (CSY128), and sec65-1Δegd2 (YNY1) strains were grown in YP media at 24°C or shifted to 37°C for 120 min to mid log phase. Lysates were extracted with nonionic detergent to yield a pellet enriched in detergent-insoluble aggregates, and analyzed by SDS-PAGE and Coomassie staining or blotting for Pho8. The total amount of aggregates quantified (error bars = SEM; n = 3; *, P < 0.05; **, P < 0.01 one-way ANOVA). (B) WT (W303) and sec65-1 (CSY128) strains were grown in YP media to mid-log phase and shifted to 37°C, and aggregates were isolated as in A. Where indicated, extracts were treated with PNGase F. Reactions were analyzed by blotting for Pho8. Position of nonglycosylated (pPho8p) and glycosylated Pho8 (g-Pho8p) is indicated. (C) WT (W303), sec65-1 (CSY128), and sec65-1Δegd2 (YNY1) cells were grown to mid-log phase in minimal media at 30°C and then pulse-labeled with [35S] methionine and cysteine for 5 min before analysis as in A but with SDS-PAGE and phosphorimaging. (D) WT (W303) and sec65-1 (CSY128) harboring CEN or 2 µm plasmids for expressing EGD1 (pMP300), EGD2 (pMP301 or pMP302), or NAC (EGD1 and EGD2 -pMP303 and pMP304) were analyzed for aggregates as in A. (E) Aggregates from WT, sec65-1 (CSY128), sec65-1Δegd2 (YNY1) and sec65-1 with a CEN EGD2 plasmid (pMP301) strains were analyzed by blotting for ubiquitin.
Figure 7.
Figure 7.
Model indicating a novel role for NAC in regulating the access of MAP and SRP to the ribosome. Short nascent chains first encounter NAC, which interacts with the ribosome close to Rpl25/35 and Rpl31/17 (1). The presence of NAC allows both SRP and Map1 to bind the ribosome and sample nascent chains (2). The presence of an SRP substrate changes the conformation of NAC, possibly in response to a signal sequence/anchor in the exit tunnel such that SRP can bind the signal sequence (3). The α subunit of NAC can prevent aggregation of the signal sequence before the arrival of SRP. Handover of the signal sequence to SRP releases NAC leading to tight binding of SRP (4). Nonsecretory proteins interact with RAC and Ssb1/2 as the nascent chain extends (5). In the absence of NAC, SRP binding precludes Map1 binding thereby compromising processing and folding of cytosolic proteins.
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