Immunoaffinity purification of endogenous proteins from S. cerevisiae for post-translational modification and protein interaction analysis.

Protein regulation by post-translational modifications and protein-protein interactions is critical to controlling molecular pathways. Here, we describe an immunoaffinity purification approach in Saccharomyces cerevisiae. The protocol uses an endogenously-expressed epitope-tagged protein and can be applied to the identification of post-translational modifications or protein binding partners. The lysine methyltransferase Set5 is used as an example here to purify phosphorylated Set5 and identify phosphosites; however, this approach can be applied to a diverse set of proteins in yeast. For complete details on the use and execution of this protocol, please refer to Jaiswal et al. (2020).

Mapping protein interactions in the active TOM-TIM23 supercomplex.

Nuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes. The TOM and TIM23 complexes facilitate the transport of precursor proteins with N-terminal targeting signals into the matrix. During transport, precursors are recognized by the TIM23 complex in the inner membrane for handover from the TOM complex. However, we have little knowledge on the organization of the TOM-TIM23 transition zone and on how precursor transfer between the translocases occurs. Here, we have designed a precursor protein that is stalled during matrix transport in a TOM-TIM23-spanning manner and enables purification of the translocation intermediate. Combining chemical cross-linking with mass spectrometric analyses and structural modeling allows us to map the molecular environment of the intermembrane space interface of TOM and TIM23 as well as the import motor interactions with amino acid resolution. Our analyses provide a framework for understanding presequence handover and translocation during matrix protein transport.

Pathway of Hsp70 interactions at the ribosome.

In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70 Ssb, atypical Hsp70 Ssz1 and J-domain protein cochaperone Zuo1. Zuo1 binds the ribosome at the tunnel exit. Zuo1 also binds Ssz1, tethering it to the ribosome, while its J-domain stimulates Ssb's ATPase activity to drive efficient nascent chain interaction. But the function of Ssz1 and how Ssb engages at the ribosome are not well understood. Employing in vivo site-specific crosslinking, we found that Ssb(ATP) heterodimerizes with Ssz1. Ssb, in a manner consistent with the ADP conformation, also crosslinks to ribosomal proteins across the tunnel exit from Zuo1. These two modes of Hsp70 Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz1, allowing optimal J-domain and nascent chain engagement; then, after ATP hydrolysis, Ssb(ADP) directly with the ribosome.

Ruler elements in chromatin remodelers set nucleosome array spacing and phasing.

Arrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP-dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP-dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the 'ruler' that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements.

Genome information processing by the INO80 chromatin remodeler positions nucleosomes.

The fundamental molecular determinants by which ATP-dependent chromatin remodelers organize nucleosomes across eukaryotic genomes remain largely elusive. Here, chromatin reconstitutions on physiological, whole-genome templates reveal how remodelers read and translate genomic information into nucleosome positions. Using the yeast genome and the multi-subunit INO80 remodeler as a paradigm, we identify DNA shape/mechanics encoded signature motifs as sufficient for nucleosome positioning and distinct from known DNA sequence preferences of histones. INO80 processes such information through an allosteric interplay between its core- and Arp8-modules that probes mechanical properties of nucleosomal and linker DNA. At promoters, INO80 integrates this readout of DNA shape/mechanics with a readout of co-evolved sequence motifs via interaction with general regulatory factors bound to these motifs. Our findings establish a molecular mechanism for robust and yet adjustable +1 nucleosome positioning and, more generally, remodelers as information processing hubs that enable active organization and allosteric regulation of the first level of chromatin.

Impact of DNA methylation on 3D genome structure.

Determining the effect of DNA methylation on chromatin structure and function in higher organisms is challenging due to the extreme complexity of epigenetic regulation. We studied a simpler model system, budding yeast, that lacks DNA methylation machinery making it a perfect model system to study the intrinsic role of DNA methylation in chromatin structure and function. We expressed the murine DNA methyltransferases in Saccharomyces cerevisiae and analyzed the correlation between DNA methylation, nucleosome positioning, gene expression and 3D genome organization. Despite lacking the machinery for positioning and reading methylation marks, induced DNA methylation follows a conserved pattern with low methylation levels at the 5' end of the gene increasing gradually toward the 3' end, with concentration of methylated DNA in linkers and nucleosome free regions, and with actively expressed genes showing low and high levels of methylation at transcription start and terminating sites respectively, mimicking the patterns seen in mammals. We also see that DNA methylation increases chromatin condensation in peri-centromeric regions, decreases overall DNA flexibility, and favors the heterochromatin state. Taken together, these results demonstrate that methylation intrinsically modulates chromatin structure and function even in the absence of cellular machinery evolved to recognize and process the methylation signal.

Crystal structures of an E1-E2-ubiquitin thioester mimetic reveal molecular mechanisms of transthioesterification.

E1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1-E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1-E2-Ub complex during catalysis are unknown. Here, we determine the structure of a covalently trapped E1-E2-ubiquitin thioester mimetic. Two distinct architectures of the complex are observed, one in which the Ub thioester (Ub(t)) contacts E1 in an open conformation and another in which Ub(t) instead contacts E2 in a drastically different, closed conformation. Altogether our structural and biochemical data suggest that these two conformational states represent snapshots of the E1-E2-Ub complex pre- and post-thioester transfer, and are consistent with a model in which catalysis is enhanced by a Ub(t)-mediated affinity switch that drives the reaction forward by promoting productive complex formation or product release depending on the conformational state.

Ribosome-bound Get4/5 facilitates the capture of tail-anchored proteins by Sgt2 in yeast.

The guided entry of tail-anchored proteins (GET) pathway assists in the posttranslational delivery of tail-anchored proteins, containing a single C-terminal transmembrane domain, to the ER. Here we uncover how the yeast GET pathway component Get4/5 facilitates capture of tail-anchored proteins by Sgt2, which interacts with tail-anchors and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of tail-anchored proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting. Exposure of internal transmembrane domains at the tunnel exit induces high-affinity ribosome binding of SRP, which in turn prevents ribosome binding of Get4/5. In this way, the position of a transmembrane domain within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit.

The biochemical and genetic discovery of the SAGA complex.

One of the major advances in our understanding of gene regulation in eukaryotes was the discovery of factors that regulate transcription by controlling chromatin structure. Prominent among these discoveries was the demonstration that Gcn5 is a histone acetyltransferase, establishing a direct connection between transcriptional activation and histone acetylation. This breakthrough was soon followed by the purification of a protein complex that contains Gcn5, the SAGA complex. In this article, we review the early genetic and biochemical experiments that led to the discovery of SAGA and the elucidation of its multiple activities.

Catalysis by protein acetyltransferase Gcn5.

Gcn5 serves as the defining member of the Gcn5-related N-acetyltransferase (GNAT) superfamily of proteins that display a common structural fold and catalytic mechanism involving the transfer of the acyl-group, primarily acetyl-, from CoA to an acceptor nucleophile. In the case of Gcn5, the target is the ε-amino group of lysine primarily on histones. Over the years, studies on Gcn5 structure-function have often formed the basis by which we understand the complex activities and regulation of the entire protein acetyltransferase family. It is now appreciated that protein acetylation occurs on thousands of proteins and can reversibly regulate the function of many cellular processes. In this review, we provide an overview of our fundamental understanding of catalysis, regulation of activity and substrate selection, and inhibitor development for this archetypal acetyltransferase.

TORC1 Determines Fab1 Lipid Kinase Function at Signaling Endosomes and Vacuoles.

Organelles of the endomembrane system maintain their identity and integrity during growth or stress conditions by homeostatic mechanisms that regulate membrane flux and biogenesis. At lysosomes and endosomes, the Fab1 lipid kinase complex and the nutrient-regulated target of rapamycin complex 1 (TORC1) control the integrity of the endolysosomal homeostasis and cellular metabolism. Both complexes are functionally connected as Fab1-dependent generation of PI(3,5)P2 supports TORC1 activity. Here, we identify Fab1 as a target of TORC1 on signaling endosomes, which are distinct from multivesicular bodies, and provide mechanistic insight into their crosstalk. Accordingly, TORC1 can phosphorylate Fab1 proximal to its PI3P-interacting FYVE domain, which causes Fab1 to shift to signaling endosomes, where it generates PI(3,5)P2. This, in turn, regulates (1) vacuole morphology, (2) recruitment of TORC1 and the TORC1-regulatory Rag GTPase-containing EGO complex to signaling endosomes, and (3) TORC1 activity. Thus, our study unravels a regulatory feedback loop between TORC1 and the Fab1 complex that controls signaling at endolysosomes.

The Proteomic Landscape of Centromeric Chromatin Reveals an Essential Role for the Ctf19CCAN Complex in Meiotic Kinetochore Assembly.

Kinetochores direct chromosome segregation in mitosis and meiosis. Faithful gamete formation through meiosis requires that kinetochores take on new functions that impact homolog pairing, recombination, and the orientation of kinetochore attachment to microtubules in meiosis I. Using an unbiased proteomics pipeline, we determined the composition of centromeric chromatin and kinetochores at distinct cell-cycle stages, revealing extensive reorganization of kinetochores during meiosis. The data uncover a network of meiotic chromosome axis and recombination proteins that bind to centromeres in the absence of the microtubule-binding outer kinetochore sub-complexes during meiotic prophase. We show that the Ctf19cCCAN inner kinetochore complex is essential for kinetochore organization in meiosis. Our functional analyses identify a Ctf19cCCAN-dependent kinetochore assembly pathway that is dispensable for mitotic growth but becomes critical upon meiotic entry. Therefore, changes in kinetochore composition and a distinct assembly pathway specialize meiotic kinetochores for successful gametogenesis.

The yeast mitophagy receptor Atg32 is ubiquitinated and degraded by the proteasome.

Mitophagy, the process that degrades mitochondria selectively through autophagy, is involved in the quality control of mitochondria in cells grown under respiratory conditions. In yeast, the presence of the Atg32 protein on the outer mitochondrial membrane allows for the recognition and targeting of superfluous or damaged mitochondria for degradation. Post-translational modifications such as phosphorylation are crucial for the execution of mitophagy. In our study we monitor the stability of Atg32 protein in the yeast S. cerevisiae and show that Atg32 is degraded under normal growth conditions, upon starvation or rapamycin treatment. The Atg32 turnover can be prevented by inhibition of the proteasome activity, suggesting that Atg32 is also ubiquitinated. Mass spectrometry analysis of purified Atg32 protein revealed that at least lysine residue in position 282 is ubiquitinated. Interestingly, the replacement of lysine 282 with alanine impaired Atg32 degradation only partially in the course of cell growth, suggesting that additional lysine residues on Atg32 might also be ubiquitinated. Our results provide the foundation to further elucidate the physiological significance of Atg32 turnover and the interplay between mitophagy and the proteasome.

Structure of the TFIIIC subcomplex τA provides insights into RNA polymerase III pre-initiation complex formation.

Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative-stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.

Isolation and Analysis of Mitochondrial Fission Enzyme DNM1 from Saccharomyces cerevisiae.

Mitochondrial fission, an essential process for mitochondrial and cellular homeostasis, is accomplished by evolutionarily conserved members of the dynamin superfamily of large GTPases. These enzymes couple the hydrolysis of guanosine triphosphate to the mechanical work of membrane remodeling that ultimately leads to membrane scission. The importance of mitochondrial dynamins is exemplified by mutations in the human family member that causes neonatal lethality. In this chapter, we describe the subcloning, purification, and preliminary characterization of the budding yeast mitochondrial dynamin, DNM1, from Saccharomyces cerevisiae, which is the first mitochondrial dynamin isolated from native sources. The yeast-purified enzyme exhibits assembly-stimulated hydrolysis of GTP similar to other fission dynamins, but differs from the enzyme isolated from non-native sources.

Reduction of the P5A-ATPase Spf1p phosphoenzyme by a Ca2+-dependent phosphatase.

P5 ATPases are eukaryotic pumps important for cellular metal ion, lipid and protein homeostasis; however, their transported substrate, if any, remains to be identified. Ca2+ was proposed to act as a ligand of P5 ATPases because it decreases the level of phosphoenzyme of the Spf1p P5A ATPase from Saccharomyces cerevisiae. Repeating previous purification protocols, we obtained a purified preparation of Spf1p that was close to homogeneity and exhibited ATP hydrolytic activity that was stimulated by the addition of CaCl2. Strikingly, a preparation of a catalytically dead mutant Spf1p (D487N) also exhibited Ca2+-dependent ATP hydrolytic activity. These results indicated that the Spf1p preparation contained a co-purifying protein capable of hydrolyzing ATP at a high rate. The activity was likely due to a phosphatase, since the protein i) was highly active when pNPP was used as substrate, ii) required Ca2+ or Zn2+ for activity, and iii) was strongly inhibited by molybdate, beryllium and other phosphatase substrates. Mass spectrometry identified the phosphatase Pho8p as a contaminant of the Spf1p preparation. Modification of the purification procedure led to a contaminant-free Spf1p preparation that was neither stimulated by Ca2+ nor inhibited by EGTA or molybdate. The phosphoenzyme levels of a contaminant-free Spf1p preparation were not affected by Ca2+. These results indicate that the reported effects of Ca2+ on Spf1p do not reflect the intrinsic properties of Spf1p but are mediated by the activity of the accompanying phosphatase.

Mannoproteins from inactivated whole cells of baker's and brewer's yeasts as functional food ingredients: Isolation and optimization.

Because of the structural multiplicity of yeast mannoproteins, they have shown a great interest as food ingredients for a wide range of applications. The yields and the structural properties of mannoproteins varied depending on the isolation methods and their sources (baker's and brewer's Saccharomyces cerevisiae yeasts). Noncovalently bound mannoproteins (6.5 kDa) with a mannan/protein ratio of 0.63 and 2.78 were recovered upon the heat treatment of brewer's and baker's yeasts, respectively, whereas sodium dodecyl sulfate treatment led mainly to the release of nonglycosylated proteins. The highest yield of mannoproteins was achieved upon the enzymatic isolation with Zymolyase® from Arthrobacter luteus. The recovered covalently bound mannoproteins were characterized by a higher mannan/protein ratio (13.1 to 42.7) and a wider molecular weight distribution (5 to 10 kDa; 10 to 100 kDa; 100 to 400 kDa). Predictive models were developed to understand and modulate the effects of isolation parameters on yield, the mannoproteins content, and the mannan/protein ratio. The enzyme concentration was the most significant parameter affecting the yield, whereas the reaction time was the most significant parameter affecting mannan/protein ratio. Comparison of predicted and experimental values validated the established predicted models for the isolation of well-defined mannoproteins from yeast for targeted food applications. PRACTICAL APPLICATION: The increasing demand for clean label health-promoting foods has fueled the development of highly functional ingredients that offer both techno-functionalities and health-promoting properties. This study reveals the efficiency of whole inactivated yeasts as sources of mannoproteins. Given the dependence of the techno-functional and health-promoting properties of mannoproteins on their molecular properties, the investigation of the effects of the yeast sources and the type of isolation methods on the structural properties of mannoproteins would allow the modulation of their properties. Furthermore, the developed predictive models for the enzymatic process are expected to enhance the isolation efficiency of mannoproteins with well-defined structures.

Expression and Purification of Membrane Proteins in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is one of the most popular expression systems for eukaryotic membrane proteins. Here, we describe protocols for the expression and purification of mitochondrial membrane proteins developed in our laboratory during the last 15 years. To optimize their expression in a functional form, different promoter systems as well as codon-optimization and complementation strategies were established. Purification approaches were developed which remove the membrane protein from the affinity column by specific proteolytic cleavage rather than by elution. This strategy has several important advantages, most notably improving the purity of the sample, as contaminants stay bound to the column, thus eliminating the need for a secondary purification step, such as size exclusion chromatography. This strategy also avoids dilution of the sample, which would occur as a consequence of elution, precluding the need for concentration steps, and thus preventing detergent concentration.

Targeted Identification of Protein Interactions in Eukaryotic mRNA Translation.

To identify protein-protein interactions and phosphorylated amino acid sites in eukaryotic mRNA translation, replicate TAP-MudPIT and control experiments are performed targeting Saccharomyces cerevisiae genes previously implicated in eukaryotic mRNA translation by their genetic and/or functional roles in translation initiation, elongation, termination, or interactions with ribosomal complexes. Replicate tandem affinity purifications of each targeted yeast TAP-tagged mRNA translation protein coupled with multidimensional liquid chromatography and tandem mass spectrometry analysis are used to identify and quantify copurifying proteins. To improve sensitivity and minimize spurious, nonspecific interactions, a novel cross-validation approach is employed to identify the most statistically significant protein-protein interactions. Using experimental and computational strategies discussed herein, the previously described protein composition of the canonical eukaryotic mRNA translation initiation, elongation, and termination complexes is calculated. In addition, statistically significant unpublished protein interactions and phosphorylation sites for S. cerevisiae's mRNA translation proteins and complexes are identified.

Genetically inspired in vitro reconstitution of Saccharomyces cerevisiae actin cables from seven purified proteins.

A major goal of synthetic biology is to define the minimal cellular machinery required to assemble a biological structure in its simplest form. Here, we focused on Saccharomyces cerevisiae actin cables, which provide polarized tracks for intracellular transport and maintain defined lengths while continuously undergoing rapid assembly and turnover. Guided by the genetic requirements for proper cable assembly and dynamics, we show that seven evolutionarily conserved S. cerevisiae proteins (actin, formin, profilin, tropomyosin, capping protein, cofilin, and AIP1) are sufficient to reconstitute the formation of cables that undergo polarized turnover and maintain steady-state lengths similar to actin cables in vivo. Further, the removal of individual proteins from this simple in vitro reconstitution system leads to cable defects that closely approximate in vivo cable phenotypes caused by disrupting the corresponding genes. Thus, a limited set of molecular components is capable of self-organizing into dynamic, micron-scale actin structures with features similar to cables in living cells.