A Genome-Wide Immunodetection Screen in S. cerevisiae Uncovers Novel Genes Involved in Lysosomal Vacuole Function and Morphology

Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface – ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY). Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes – MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events

The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene

The S. cerevisiae VPS10 (vacuolar protein sorting) gene encodes a type I transmembrane protein of 1577 amino acids required for the sorting of the soluble vacuolar protein carboxypeptidase Y (CPY). Mutations in VPS10 result in the selective missorting and secretion of CPY; all other vacuolar proteins tested are delivered to the vacuole in vps10 mutants. Chemical cross-linking studies demonstrate that Vps10p and the Golgi-modified precursor form of CPY directly interact. A single amino acid change in the CPY vacuolar sorting signal prevents this interaction. Vps10p also interacts with a hybrid protein containing the CPY sorting signal fused to the normally secreted enzyme invertase. Subcellular fractionation indicates that the majority of Vps10p is localized to a late Golgi compartment where vacuolar proteins are sorted. We propose that VPS10 encodes a CPY sorting receptor that executes multiple rounds of sorting by cycling between the late Golgi and a prevacuolar endosome-like compartment

<i>ENV7</i>, <i>ENV9</i>, <i>ENV10</i>, <i>ENV11</i> ORF's complement <i>env</i> phenotype of their corresponding deletion mutants.

<p>The four strains deleted in each of the orphan genes were transformed with parent CEN vector or CEN vector containing the corresponding deleted ORF and its flanking 300–500 upstream and downstream sequences. Transformed cells were patched onto SM-URA plates for selection and were subjected to colony immunodetection with anti-proCPY specific mAb. WT(BY4742) transformed with parent or recombinant vectors containing each of the four ORF's were grown and processed in parallel as controls.</p

Steady-state accumulation of p2CPY in <i>env</i> mutants.

<p>Strains were grown in YPD overnight and were then diluted for further growth to logarithmic phase in YPD (A), or YPD+0.75 M NaCl (B). Cell extracts were normalized for protein content and subjected to western analysis. <i>pep4Δ</i> mutants are completely defective in CPY processing and a 1∶5 dilution of cell lysate was used as control for more equivalent visualization of signal. Membranes were probed with anti-proCPY mAb; hexokinase I was used as loading control and detected with pAb's. Two separate gels in A are marked by a vertical bar.</p

Genomic immunodetection screen- flowchart and results.

<p><b>A.</b> Flowchart of screen strategy and results as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023696#s4" target="_blank">Materials and Methods</a> section. <b>B.</b> Repeated immunodetections with mAb specific to pro region of CPY confirmed twelve mutants that internally accumulate pro-CPY (<i>env</i> mutants) and one new pro-CPY secreting mutant (<i>vps</i> mutant), <i>ecm27Δ</i>. The BY4742 (WT), <i>pep4Δ</i> and <i>vps35Δ</i> strains are included as controls (L = lysed, UL = unlysed).</p

Growth characterizations of <i>env</i> mutants under various ion and drug conditions.

<p>Equivalent OD₆₀₀ units of WT (BY4742) and mutants were stamped at multiple dilutions as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023696#s4" target="_blank">Materials and Methods</a>. Wild type, <i>env7Δ</i> and <i>env9Δ</i> strains are represented in the figure; data for all mutants are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023696#pone-0023696-t002" target="_blank">Table 2</a>.</p

Growth characterizations of uncovered mutants under selective conditions.

<p>Growth characterizations of uncovered mutants under selective conditions.</p

Vacuolar morphology of uncovered mutants.

<p><b>A.</b> Logarithmically growing cells were stained with vital dye FM4-64 for 60 min and viewed using DIC optics and confocal microscopy. The predominant vacuolar morphology observed is shown. <b>B.</b> Percentages of observed vacuolar morphology phenotypes. For each strain, 300–400 non-budding cells were scored in randomized fields; raw data were subjected to Chi-squared analysis. Mutants showing statistically significant difference from wild type are in bold. <b>C.</b> Strains exhibiting abnormal vacuolar morphology were subject to costaining with vital dyes FM4-64 and CellTracker Blue CMAC. <b>D.</b> Representative DIC and fluorescent confocal images of FM4-64 stained <i>env9Δ.</i></p

Invertase and active α-factor secretion in <i>env</i> mutants.

<p><b>A.</b> A quantitative colorimetric assay for glucose presence was used to assess invertase secretion as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023696#s2" target="_blank">Results</a>. Secreted invertase is expressed as the percentage of extracellular invertase over total intracellular and extracellular .invertase. Bars indicate standard deviation (Two-Sample <i>t</i>-Test, N = 3, confidence interval 95%, <i>p</i><0.05). <b>B.</b> Qualitative halo assays were used to assess active α-factor secretion by equivalent inocula of MAT-α <i>env</i> strains and isogenic WT onto a lawn of supersensitive MAT-a GPY-1796 cells. Each vertical column is a single plate with its WT control; visual alignment was achieved using Photoshop without alteration of experimental data.</p