Functional Dissection of the Nascent Polypeptide-Associated Complex in Saccharomyces cerevisiae - PubMed (original) (raw)

Functional Dissection of the Nascent Polypeptide-Associated Complex in Saccharomyces cerevisiae

Ann-Kathrin Ott et al. PLoS One. 2015.

Abstract

Both the yeast nascent polypeptide-associated complex (NAC) and the Hsp40/70-based chaperone system RAC-Ssb are systems tethered to the ribosome to assist cotranslational processes such as folding of nascent polypeptides. While loss of NAC does not cause phenotypic changes in yeast, the simultaneous deletion of genes coding for NAC and the chaperone Ssb (nacΔssbΔ) leads to strongly aggravated defects compared to cells lacking only Ssb, including impaired growth on plates containing L-canavanine or hygromycin B, aggregation of newly synthesized proteins and a reduced translational activity due to ribosome biogenesis defects. In this study, we dissected the functional properties of the individual NAC-subunits (α-NAC, β-NAC and β'-NAC) and of different NAC heterodimers found in yeast (αβ-NAC and αβ'-NAC) by analyzing their capability to complement the pleiotropic phenotype of nacΔssbΔ cells. We show that the abundant heterodimer αβ-NAC but not its paralogue αβ'-NAC is able to suppress all phenotypic defects of nacΔssbΔ cells including global protein aggregation as well as translation and growth deficiencies. This suggests that αβ-NAC and αβ'-NAC are functionally distinct from each other. The function of αβ-NAC strictly depends on its ribosome association and on its high level of expression. Expression of individual β-NAC, β'-NAC or α-NAC subunits as well as αβ'-NAC ameliorated protein aggregation in nacΔssbΔ cells to different extents while only β-NAC was able to restore growth defects suggesting chaperoning activities for β-NAC sufficient to decrease the sensitivity of nacΔssbΔ cells against L-canavanine or hygromycin B. Interestingly, deletion of the ubiquitin-associated (UBA)-domain of the α-NAC subunit strongly enhanced the aggregation preventing activity of αβ-NAC pointing to a negative regulatory role of this domain for the NAC chaperone activity in vivo.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Ribosome-associated chaperones from S. cerevisiae.

a) The Hsp70/Hsp40-chaperone system that consists of RAC (Ssz and Zuo), shown in purple and light green, and Ssb, shown in light blue, forms a functional triad at the ribosome. In addition, β-NAC (shown in blue) and α-NAC (shown in red) that contains a C-terminal UBA (ubiquitin-associated) domain constitute the stable heterodimeric αβ-NAC complex which binds to the ribosome via the ribosome-binding motif in the β-subunit. Both, NAC and Ssb can interact directly with the nascent chain. b) Schematic representation of the different NAC subunits. α-NAC (shown in red) contains a NAC domain and a UBA domain. Besides the NAC domain the two different β-subunits (shown in light and dark blue) also contain a conserved ribosome-binding motif present in their N-termini. c) Schematic drawing of the two NAC mutants investigated in this study. αΔUBA-NAC (shown in red) lacks the C-terminal UBA domain and part of the linker region. In βRRK/AAA-NAC (shown in blue) the conserved RRK-(X)2-KK motif was mutated to AAA-(X)2-KK to abolish ribosome binding.

Fig 2

Fig 2. The αβ-NAC complex and β-NAC under control of their endogenous promoter complement the growth defect of nacΔssbΔ cells.

a) Schematic drawing of the different plasmid-encoded NAC constructs used in this study. Plasmids encoding wild type (wt) and mutant αβ-NAC, either alone or in complex, were cloned in the vector backbone pRS316 reported by [18]. b) Growth analysis of wt and mutant yeast cells expressing different NAC versions from plasmids as indicated. Serial dilutions were spotted on synthetic complete media without uracil (SD-Ura) containing the indicated drugs. When cells were plated on the arginine analogue L-canavanine, arginine was omitted. The cells were incubated for 3 days at 30°C. c) The promoter (P)—and terminator (T)- regions of EGD1 were replaced with the corresponding regions of BTT1 and vice versa and cloned in the vector backbone of pRS316 with or without EGD2. BTT1 under its endogenous promoter and terminator was also cloned into pRS316 together with EGD2. d) Growth analyses were performed as described in b).

Fig 3

Fig 3. Halfmer formation of nacΔssbΔ knockout cells can be prevented by expression of αβ-NAC.

a-i) Polysome profiles derived from wild type (wt) or mutant yeast cells. Absorbance traces at 254 nm are shown. Cells were grown to an optical density (OD600) of 0.8 in SD-Ura medium. 10 A260 units of lysates of indicated cells were loaded onto 15–45% linear sucrose gradients to isolate ribosomal fractions (40S, 60S, 80S and polysomes) as indicated by centrifugation and subsequent fractionation. Polysome profiles show: a) wt + empty vector (ev), b) ssbΔ cells + ev, c-i) nacΔssbΔ cells + ev (c), + β-NAC (d), β’-NAC (e), α ΔUBA β-NAC (f), αβ-NAC (g) + αβ’-NAC (h) and αβ RRK/AAA -NAC (i) The profiles are representative for three independent experiments.

Fig 4

Fig 4. Expression levels of β-NAC are important for complementation of nacΔssbΔ halfmers.

Polysome profiling with wild type (wt) or mutant yeast cells. Absorbance traces at 254 nm are shown. 10 A260 units of lysates of indicated yeast cells were loaded onto 15–45% linear sucrose gradients similar to Fig 3. a) Polysome profile of nacΔssbΔ cells + empty vector control (ev) (in grey). b) and c) Complementation of nacΔssbΔ cells with promoter-swapped β-NAC constructs alone. d) nacΔssbΔ cells expressing α-NAC alone. e) and f) Polysome profiles of nacΔssbΔ cells expressing promoter-swapped β-NAC constructs in combination with α-NAC. Arrows indicate halfmers. The profiles are representative for three independent runs.

Fig 5

Fig 5. NAC is not coregulated with genes encoding ribosomal proteins.

a) X-axis: Relative mRNA levels of indicated genes and time points compared to timepoint zero (t = 0, before glucose addition) and normalized to an internal control (housekeeping gene). Cells were harvested at 0 min, 30 min and 60 min after glucose addition and mRNA was extracted. cDNA was obtained by reverse transcription and used for qRT-PCR. b) Serial dilutions of wild type (wt) and chaperone mutant cells were spotted on YPD plates and plates containing the indicated drugs for growth analysis. When cells were plated on the arginine analogue L-canavanine, arginine was omitted. The cells were incubated for 3 days at 30°C. c) Polysome profiles of wt and mutant cells. 10 A260 units of lysates of indicated yeast strains were loaded onto 15–45% linear sucrose gradients as shown in Fig 3. The profiles are representative for three independent runs.

Fig 6

Fig 6. Analysis of protein aggregation in nacΔssbΔ suppressed by NAC variants.

a) 50 OD600 units of transformed yeast cells expressing the indicated NAC variants in the logarithmic phase were lysed and the aggregated protein material was isolated by sedimentation. Isolated aggregated fractions were separated by SDS-PAGE and visualized by Coomassie staining. b) Biological replicates of the experiment shown in a) for aggregated proteins of wt, ssbΔ and nacΔssbΔ cells (lanes 1–3), nacΔssbΔ + αβ-NAC (lane 8) and + α ΔUBA β-NAC (lane 10). The experiment was performed as in a). For better visualization the corresponding lanes were cut out from the same SDS-PAGE after Coomassie staining as indicated by black lines. c) Quantification of aggregated material using ImageJ shows the relative level of aggregated protein in relation to total protein amount, normalized to the mean value of wt replicates. Mean ± SD is shown from three experiments (n = 3).

Fig 7

Fig 7. β-NAC and β’-NAC show differences in their C-termini.

First, a PSI-BLAST search of the NCBI database was performed. Then the sequences were sorted using CLANS [34] and aligned with the alignment programme muscle [35]. An HMM (

http://hmmer.org/

) was constructed of the fungi sequences and all sequences were aligned against the HMM. The sequences are shown for a) the N-terminus, b) the NAC-domain and c) the C-terminus of β-NAC and β’-NAC of S. cerevisiae, and of β-NAC from C. elegans and H. sapiens. Amino acids are depicted in the one letter-code. β-NAC of S. cerevisiae could be aligned completely to the β-sequences of all kingdoms, but the end of the C-terminus of β’ from S. cerevisiae could not be aligned with the other sequences and was marked as an insert (small letters at the end of the alignment). Colour legend: orange = small hydrophilics, green = small hydrophobics, red = bases, blue = aromatics and colourless = acids/amides and sulphhydrils.

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This work was supported by a research grant from the German Science Foundation (DFG; SFB969/A01) to ED.

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