Functional Dissection of the Nascent Polypeptide-Associated Complex in <i>Saccharomyces cerevisiae</i>

<div><p>Both the yeast <b>n</b>ascent polypeptide-<b>a</b>ssociated <b>c</b>omplex (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 (<i>nacΔssbΔ</i>) 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 <i>nacΔssbΔ</i> cells. We show that the abundant heterodimer αβ-NAC but not its paralogue αβ’-NAC is able to suppress all phenotypic defects of <i>nacΔssbΔ</i> 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 <i>nacΔssbΔ</i> 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 <i>nacΔssbΔ</i> cells against L-canavanine or hygromycin B. Interestingly, deletion of the <b>ub</b>iquitin-<b>a</b>ssociated (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 <i>in vivo</i>.</p></div

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

<p>First, a PSI-BLAST search of the NCBI database was performed. Then the sequences were sorted using CLANS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143457#pone.0143457.ref034" target="_blank">34</a>] and aligned with the alignment programme muscle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143457#pone.0143457.ref035" target="_blank">35</a>]. An HMM (<a href="http://hmmer.org/" target="_blank">http://hmmer.org/</a>) 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 <i>S</i>. <i>cerevisiae</i>, and of β-NAC from <i>C</i>. <i>elegans</i> and <i>H</i>. <i>sapiens</i>. Amino acids are depicted in the one letter-code. β-NAC of <i>S</i>. <i>cerevisiae</i> could be aligned completely to the β-sequences of all kingdoms, but the end of the C-terminus of β’ from <i>S</i>. <i>cerevisiae</i> 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.</p

<i>NAC</i> is not coregulated with genes encoding ribosomal proteins.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143457#pone.0143457.g003" target="_blank">Fig 3</a>. The profiles are representative for three independent runs.</p

Ribosome-associated chaperones from <i>S</i>. <i>cerevisiae</i>.

<p>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. α^ΔUBA-NAC (shown in red) lacks the C-terminal UBA domain and part of the linker region. In β^RRK/AAA-NAC (shown in blue) the conserved RRK-(X)₂-KK motif was mutated to AAA-(X)₂-KK to abolish ribosome binding.</p

Expression levels of <i>β-NAC</i> are important for complementation of <i>nacΔssbΔ</i> halfmers.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143457#pone.0143457.g003" target="_blank">Fig 3</a>. a) Polysome profile of <i>nacΔssbΔ</i> cells + empty vector control (ev) (in grey). b) and c) Complementation of <i>nacΔssbΔ</i> cells with promoter-swapped <i>β-NAC</i> constructs alone. d) <i>nacΔssbΔ</i> cells expressing α-NAC alone. e) and f) Polysome profiles of <i>nacΔssbΔ</i> cells expressing promoter-swapped β-NAC constructs in combination with α-NAC. Arrows indicate halfmers. The profiles are representative for three independent runs.</p

The αβ-NAC complex and β-NAC under control of their endogenous promoter complement the growth defect of <i>nacΔssbΔ</i> cells.

<p>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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143457#pone.0143457.ref018" target="_blank">18</a>]. 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 <i>EGD1</i> were replaced with the corresponding regions of <i>BTT1</i> and <i>vice versa</i> and cloned in the vector backbone of pRS316 with or without <i>EGD2</i>. <i>BTT1</i> under its endogenous promoter and terminator was also cloned into pRS316 together with <i>EGD2</i>. d) Growth analyses were performed as described in b).</p

Halfmer formation of <i>nacΔssbΔ</i> knockout cells can be prevented by expression of <i>αβ-NAC</i>.

<p>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) <i>ssbΔ</i> cells + ev, c-i) <i>nacΔssbΔ</i> cells + ev (c), + <i>β-NAC</i> (d), <i>β’-NAC</i> (e), <i>α</i>^<i>ΔUBA</i><i>β-NAC</i> (f), <i>αβ-NAC</i> (g) + <i>αβ’-NAC</i> (h) and <i>αβ</i>^{<i>RRK/AAA</i>}<i>-NAC</i> (i) The profiles are representative for three independent experiments.</p

Analysis of protein aggregation in <i>nacΔssbΔ</i> suppressed by <i>NAC</i> variants.

<p>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, <i>ssbΔ</i> and <i>nacΔssbΔ</i> cells (lanes 1–3), <i>nacΔssbΔ</i> + <i>αβ-NAC</i> (lane 8) and + <i>α</i>^<i>ΔUBA</i><i>β-NAC</i> (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).</p