Cis1/Atg31 is required for autophagosome formation in Saccharomyces cerevisiae

Autophagy is the bulk degradation of cytosolic materials in lysosomes/vacuoles of eukaryotic cells. In the yeast Saccharomyces cerevisiae, 17 Atg proteins are known to be involved in autophagosome formation. Genome wide analyses have shown that Atg17 interacts with numerous proteins. Further studies on these interacting proteins may provide further insights into membrane dynamics during autophagy. Here, we identify Cis1/Atg31 as a protein that exhibits similar phenotypes to Atg17. ATG31 null cells were defective in autophagy and lost viability under starvation conditions. Localization of Atg31 to pre-autophagosomal structures (PAS) was dependent on Atg17. Coimmunoprecipitation experiments indicated that Atg31 interacts with Atg17. Together, Atg31 is a novel protein that, in concert with Atg17, is required for proper autophagosome formation.     

Characterization of the Atg17-Atg29-Atg31 complex specifically required for starvation-induced autophagy in Saccharomyces cerevisiae

Nutrient starvation induces autophagy to degrade cytoplasmic materials in the vacuole/lysosomes. In the yeast, Saccharomyces cerevisiae, Atg17, Atg29, and Atg31/Cis1 are specifically required for autophagosome formation by acting as a scaffold complex essential for pre-autophagosomal structure (PAS) organization. Here, we show that these proteins constitutively form an Atg17-Atg29-Atg31 ternary complex, in which phosphorylated Atg31 is included. Reconstitution analysis of the ternary complex in E. coli indicates that the three proteins are included in equimolar amounts in the complex. The molecular mass of a monomeric Atg17-Atg29-Atg31 complex is calculated at 97 kDa; however, analytical ultracentrifugation shows that the molecular mass of the ternary complex is 198 kDa, suggesting a dimeric complex. We propose that this ternary complex acts as a functional unit for autophagosome formation.     

Subcellular localization of the interaction of bipolar landmarks Bud8p and Bud9p with Rax2p in Saccharomyces cerevisiae diploid cells

In Saccharomyces cerevisiae, the bud site selection of diploid cells is regulated by at least four persistent landmarks, Bud8p, Bud9p, Rax1p, and Rax2p. Bud8p and Bud9p are essential for the establishment of bipolar budding and localize mainly to the distal and the proximal poles, respectively. Their subcellular localizations are regulated through interaction with Rax1p/Rax2p. We investigated when and where Bud8p and Bud9p physically interact with Rax2p in vivo using a split-GFP method. GFP fluorescence showed that Bud8p physically interacted with Rax2p at the proximal or distal pole in unbudded cells; a physical interaction was also observed at the opposite pole to the growing bud in mother cells with a large-size bud. Bud9p physically interacted with Rax2p at the birth scar in budded mother cells. These observations suggest that the interaction of Rax2p with Bud8p and Bud9p may contribute to the translocation of bipolar landmarks to the correct sites.     

Nocodazole induces mitotic cell death with apoptotic-like features in Saccharomyces cerevisiae

We investigated the fate of budding yeast treated with nocodazole, a microtubule-depolymerizing drug. Cells died after mitotic arrest while staying in mitosis, suggesting that mitotic cell death, but not mitotic slippage, mainly occurs in nocodazole-treated cells. Nocodazole-treated cells showed features of apoptotic-like cell death, but not those of cell lysis or autophagy. Consistently, mitochondria-dependent production of reactive oxygen species was involved in the cell death. Similar cell death was also seen in cells after mitotic arrest by perturbation of the anaphase-promoting complex/cyclosome. In addition, caspase activity was found in nocodazole-treated cells, which was independent of the metacaspase, Mca1. Our results suggest that budding yeast can be a model to study mitotic cell death in cancer treatment with antimitotic drugs.     

Characterization of five terminator regions that increase the protein yield of a transgene in Saccharomyces cerevisiae

Strong terminator regions could be used to improve metabolically engineered yeasts by increasing the target enzyme protein yields above those achieved with traditional terminator regions. We recently identified five strong terminator regions (RPL41Bt, RPL15At, DIT1t, RPL3t, and IDP1t) in a comprehensive analysis of Saccharomyces cerevisiae. The effect of the terminator regions was analyzed by measuring the protein production of a linked transgene, and was shown to be twice that of a traditional terminator region (PGK1t). Here, we investigated whether the activity of the terminator regions is affected by exchange of a strong promoter or reporter in the linked transgene, carbon source for cell growth, stress factors, host yeast strain, or stage of the growth phase. Our results indicate that the activities of all five terminator regions were twice that of PGK1t in all conditions tested. In addition, we demonstrated that the strong activity of these terminator regions could be used to improve secretory production of endoglucanase II derived from Tricoderma ressei, and that the DIT1t strain was the best of the five strains for this purpose. We therefore propose that DIT1t, and the four other terminator regions, could be applied to the development of improved metabolically engineered yeasts.     

Identification of a novel Ser/Thr protein phosphatase Ppq1 as a negative regulator of mating MAP kinase pathway in Saccharomyces cerevisiae

The specificity and efficiency of cell signaling is largely governed by the complex formation of signaling proteins. The precise spatio-temporal control of the complex assembly is crucial for proper signaling and cell survival. Protein phosphorylation is a key mechanism of signal processing in most of cell signaling networks. Phosphatases, along with kinases, control the phosphorylation state of many proteins and thus play a critical role in the precise regulation of signaling at each stage such as activation, propagation, and adaptation. Identification and functional analysis of pathway-specific phosphatase is, therefore, crucial for the understanding of cell signaling mechanisms. Here, we have developed a novel screening strategy to identify pathway-specific phosphatases, in which the entire repertoire of cell’s phosphatases was tethered to a signaling complex and the changes in signaling response were monitored. As a model target, we have chosen the mating MAP kinase pathway in the budding yeast, which is composed of three kinases and Ste5 scaffold protein. Using this strategy, a putative Ser/Thr phosphatase, Ppq1, was identified to be mating-specific. Results show that Ppq1 down-regulates mating signaling by targeting at or upstream of the terminal MAP kinase Fus3 in the cascade. The catalytic activity of Ppq1 as a phosphatase was confirmed in vitro and is necessary for its function in the regulation of mating signaling. Overall, the data suggest that Ppq1 functions as a negative regulator of mating MAPK pathway by dephosphorylating target pathway protein(s) and plays a key role in the control of the background signaling noise.     

In vivo quantification of protein-protein interactions in Saccharomyces cerevisiae using bimolecular fluorescence complementation assay

Most of the biological processes are carried out and regulated by dynamic networks of protein-protein interactions. In this study, we demonstrate the feasibility of the bimolecular fluorescence complementation (BiFC) assay for in vivo quantitative analysis of protein-protein interactions in Saccharomyces cerevisiae. We show that the BiFC assay can be used to quantify not only the amount but also the cell-to-cell variation of protein-protein interactions in S. cerevisiae. In addition, we show that protein sumoylation and condition-specific protein-protein interactions can be quantitatively analyzed by using the BiFC assay. Taken together, our results validate that the BiFC assay is a very effective method for quantitative analysis of protein-protein interactions in living yeast cells and has a great potential as a versatile tool for the study of protein function.     

Sphingolipid synthesis is involved in autophagy in Saccharomyces cerevisiae

In eukaryotes, autophagy is a conserved protein degradation system that degrades cytoplasmic components by encompassing them with double-membrane structures, called autophagosomes, and delivering them to the lytic compartments of vacuoles/lysosomes. Certain Atg proteins are known to be involved in autophagy, yet the identity and function of lipid molecules involved remain largely unknown. We investigated the involvement of sphingolipids in autophagy using Saccharomyces cerevisiae. Inhibiting synthesis of the simplest complex sphingolipid, inositol phosphorylceramide (IPC), resulted in reduced autophagic activities. Similar results were obtained using myriocin, an inhibitor of the first step in sphingolipid synthesis. Our results indicate that sphingolipids, especially IPC, are required for autophagy. Inhibition of sphingolipid synthesis had no effect on formation of Atg12-Atg5 or Atg8-phosphatidylethanolamine conjugates, on maturation of vacuolar proteases, or on formation of the pre-autophagosomal structure (PAS). These results suggest that sphingolipids are not involved in the cellular signaling that leads to formation of the PAS, but may be involved in the process of autophagosome formation.     

Chloroplast Import Signals: The Length Requirement for Translocation In Vitro and In Vivo

Protein translocation of cytosolically synthesized proteins requires signals for both targeting of precursor proteins to the surface of the respective compartment and their transfer across its membrane. In contrast to signals for peroxisomal and endoplasmic reticulum translocation, the signals for mitochondrial and chloroplast transport are less well defined with respect to length and amino acid requirements. To study the properties of signals required for translocation into chloroplasts in vitro and in vivo, we used fusion proteins composed of transit peptides and the Ig-like module of the muscle protein titin as passenger. We observed that about 60 amino acids—longer than the transit peptide length of many experimentally confirmed chloroplast proteins—are required for efficient translocation. However, within native chloroplast precursor proteins with transit peptides shorter than 60 amino acids, extension appears to be present as they are efficiently imported into organelles. In addition, the interaction of an unfolded polypeptide stretch of 60 or more amino acids with receptors at the chloroplast surface results in the unidirectionality of protein translocation into chloroplasts even in the presence of a competing C-terminal peroxisomal targeting signal. These findings prove the existing ideas that initial targeting is defined by the N-terminal signal and that the C-terminal signal is sensed only subsequently.     

Quantitative proteomic analysis of ribosomal protein L35b mutant of Saccharomyces cerevisiae

Recent studies have revealed that in higher eukaryotes, several ribosomal proteins are involved in some pathological events or developmental defects, indicating that ribosomal proteins perform unconventional functions other than protein biosynthesis. To obtain an insight into the novel roles of ribosomal proteins, we aimed to analyze the changes in proteome expression in ribosomal protein mutants by using Saccharomyces cerevisiae as a model system. We introduced the rpl35bΔ mutation into the 4159 green fluorescent protein (GFP)-tagged yeast strains by using the synthetic genetic array (SGA) method, and performed quantitative proteomic analysis by using a multilabel microplate reader and flow cytometer. We identified 22 upregulated and 20 downregulated proteins in the rpl35bΔ mutant. These proteins were primarily classified into the Gene Ontology (GO) categories of cellular biosynthetic process, translation, protein or nucleotide metabolic process, cell wall organization and biogenesis, and hyperosmotic response. We also investigated the correlation between the mRNA and protein levels of the identified proteins. Our results show that a ribosomal protein mutation can lead to perturbation in the expression of several proteins, including some other ribosomal proteins. Furthermore, our approach of combining a library of GFP-tagged yeast strains and the SGA method provides an effective and highly sensitive method for dynamic analysis of the effects of various mutations on proteome expression.     

Cdc14 Phosphatase Promotes TORC1-Regulated Autophagy in Yeast

Cdc14 protein phosphatase is critical for late mitosis progression in budding yeast, although its orthologs in other organisms, including mammalian cells, function as stress-responsive phosphatases. We found herein unexpected roles of Cdc14 in autophagy induction after nutrient starvation and target of rapamycin complex 1 (TORC1) kinase inactivation. TORC1 kinase phosphorylates Atg13 to repress autophagy under nutrient-rich conditions, but if TORC1 becomes inactive upon nutrient starvation or rapamycin treatment, Atg13 is rapidly dephosphorylated and autophagy is induced. Cdc14 phosphatase was required for optimal Atg13 dephosphorylation, pre-autophagosomal structure formation, and autophagy induction after TORC1 inactivation. In addition, Cdc14 was required for sufficient induction of ATG8 and ATG13 expression. Moreover, Cdc14 activation provoked autophagy even under normal conditions. This study identified a novel role of Cdc14 as the stress-responsive phosphatase for autophagy induction in budding yeast.     

Molecular machinery of autophagosome formation in yeast, Saccharomyces cerevisiae

Autophagy is a degradation process accompanied by dynamic membrane organization. In the yeast, Saccharomyces cerevisiae, about 30 ATG (autophagy-related) genes have been identified as important genes for autophagy. Among them, 17 are indispensable for formation of the autophagosome, an organelle enclosed by a double lipid bilayer during starvation-induced autophagy. Recently, a central structure for autophagosome generation, termed the pre-autophagosomal structure, was identified. Despite intensive study, many questions regarding the mechanisms underlying autophagosome formation remain unanswered. In this review, we will give an overview of recent studies on the mechanisms of autophagosome formation and discuss these unresolved questions.     

Nucleophosmin is required for chromosome congression, proper mitotic spindle formation, and kinetochore-microtubule attachment in HeLa cells

Nucleophosmin (NPM) is an abundantly expressed multifunctional nucleolar phosphoprotein. Here we show that depletion of NPM by RNA interference causes defects in cell division, followed by an arrest of DNA synthesis due to activation of a p53-dependent checkpoint response in HeLa cells. Depletion of NPM leads to mitotic arrest due to spindle checkpoint activation. The mitotic cells arrested by NPM depletion have defects in chromosome congression, proper mitotic spindle and centrosome formation, as well as defects in kinetochore-microtubule attachments. Loss of NPM thus causes severe mitotic defects and delayed mitotic progression. These findings indicate that NPM is essential for mitotic progression and cell proliferation.     

Cu,Zn-superoxide dismutase is necessary for proper function of VDAC in Saccharomyces cerevisiae cells

Available data suggest that a copper-and zinc-containing dismutase (CuZnSOD) plays a significant role in protecting eukaryotic cells against oxidative modifications which may contribute to cell aging. Here we demonstrated that depletion of CuZnSOD in Saccharomyces cerevisiae cells (Δsod1 cells) affected distinctly channel activity of VDAC (voltage dependent anion selective channel) and resulted in a moderate reduction in VDAC levels as well as in levels of protein crucial for VDAC import into mitochondria, namely Tob55/Sam50 and Tom40. The observed alterations may result in mitochondriopathy and subsequently in the shortening of the replicative life span observed for S. cerevisiaeΔsod1 cells.     

In vitro characterization of ligand-induced oligomerization of the S. cerevisiae G-protein coupled receptor,Ste2p

Background

The S. cerevisiae α-factor receptor, Ste2p, is a G-protein coupled receptor that plays key roles in yeast signaling and mating. Oligomerization of Ste2p has previously been shown to be important for intracellular trafficking, receptor processing and endocytosis. However the role of ligand in receptor oligomerization remains enigmatic.

Methods

Using functional recombinant forms of purified Ste2p, atomic force microscopy, dynamic light scattering and chemical crosslinking are applied to investigate the role of ligand in Ste2p oligomerization.

Results

Atomic force microscopy images indicate a molecular height for recombinant Ste2p in the presence of α-factor nearly double that of Ste2p alone. This observation is supported by complementary dynamic light scattering measurements which indicate a ligand-induced increase in the polydispersity of the Ste2p hydrodynamic radius. Finally, chemical cross-linking of HEK293 plasma membranes presenting recombinant Ste2p indicates α-factor induced stabilization of the dimeric form and higher order oligomeric forms of the receptor upon SDS-PAGE analysis.

Conclusions

α-factor induces oligomerization of Ste2p in vitro and in membrane.

General significance

These results provide additional evidence of a possible role for ligand in mediation of Ste2p oligomerization in vivo.