New & Noteworthy

Role of Sumoylation in Regulating Replication

September 09, 2022

There are ~400 origins of replication in yeast, each of which can be “licensed” by the binding of the conserved origin recognition complex (ORC) and then the MCM replicative helicase complex, all of which happens in G1 phase. During the subsequent S phase, origins are then “activated” by binding of several other replication factors, leading to unwinding and then nascent strand synthesis.

The regulation of origin licensing and activation is a complex, multi-level process, for which numerous aspects of the picture remain unclear. As yeast has the most tools for studying this process, including a full map of origins and numerous options for genetic and biochemical modulations, it presents the ideal model in which to ask probing questions. A recent study by Regan-Mochrie et al. in Genes & Development has revealed the key role of sumoylation in regulation of genome replication.

The origin recognition complex comprises six subunits, encoded by ORC1 to ORC6. The authors built on previous results showing sumoylation of these proteins during DNA damage to ask about their modification status under normal growth. They were able to assess sumoylation status for four of the six subunits, observing various degrees of sumoylation of Orc1p, Orc2p, Orc4p, and Orc5p. They then created a construct to hypersumoylate Orc2p to ask how this affects cells, and found it led to cell lethality. This lethal effect was specific to the Orc protein, as other non-Orc hypersumoylated proteins were tolerated.

The lethality could be rescued by reducing the levels of a SUMO-conjugating enzyme, further indicating the specificity of the effect. The authors identified a subset of early origins that were preferentially inhibited upon hypersumoylation and, upon study, determined that the extra sumoylation interfered with loading of the MCM complex.

After identifying the residues becoming sumoylated on Orc2p, the authors were able to generate mutants to ask about the converse, i.e. hyposumoylation. Indeed, as might be hypothesized, lack of sumoylation caused DNA replication defects via abnormal increased firing of early origins.

Thus, by close study in yeast, the role of sumoylation in genomic stability becomes more clear, where sumoylation of ORC subunits affects loading of the MCM complex, which is itself the substrate for loading of activation factors. Modulated sumoylation status appears to provide a key level of regulatory control.

Categories: Research Spotlight

Tags: DNA replication, Saccharomyces cerevisiae, cell cycle control, sumoylation, origin recognition complex, ORC complex, regulation of replication

Humanizing Glycolysis in Yeast

September 02, 2022

While glycolysis is highly conserved between unicellular yeast and multicellular humans, it appears that not only the glycolytic functions but also the secondary (aka “moonlighting”) functions of the relevant proteins remain largely consistent. This consistency presents unique opportunities for better understanding human glycolysis in muscle tissue and elsewhere.

From Boonekamp et al., 2022

The technical challenges of studying glycolytic enzymes are not trivial—largely due to genetic redundancy for such critical components—but a recent report by Boonekamp et al. in Cell Reports explains how the authors built on previous work to overcome the obstacles in yeast.  Employing a minimized set of glycolytic genes that were also relocated to a single chromosome (strain SwYG) made it possible to swap in human orthologs with relative ease.

The authors first looked at direct complementation of yeast genes by 25 human glycolytic enzymes. Remarkably, 22 of 25 human genes readily complemented their yeast counterpart. The three exceptions were hexokinases 1, 2, and 3 (HsHK1, HsHK2, and HsHK3), which are roughly twice the size of their yeast orthologs and have lower sequence conservation.

From Boonekamp et al., 2022. Numbers are percentage identities; bold indicates successful complementation.

As glycolysis cannot take place without the hexokinases, the authors first looked more closely at HsHK1 and HsHK2 to ask why they fail to function in yeast. Upon exposure to glucose, for which the native human proteins could not support growth, they created conditions to select for systematic mutations that conferred improved glycolysis. The mutant proteins proved to be less sensitive to inhibition by glucose-6-phosphate (G6P), a potent allosteric inhibitor of hexokinase activity.

With this understanding, the authors set out to complement the entire pathway in yeast with human proteins. They created two different strains, one with HsHK2 because it is considered the main isoenzyme in human muscle (strain HsGly-HK2), and one with HsHK4 because it shows less inhibition by G6P (strain HsGly-HK4). The HsGly-HK2 strain could not grow well on glucose until after a long lag phase in which mutations were selected near the G6P-binding site of HsHK2.

From Boonekamp et al., 2022. Yields on glucose (CMol/CMol) of ethanol, CO2, biomass, acetate, and glycerol are indicated (YSEthanol, YSCo2, YSX, YSAcetate, and YSglycerol, respectively).

Comparing the uptake and output of the two humanized yeast strains to the native yeast strain revealed a number of intriguing differences, especially around the different enzyme activities between human and yeast. These differences led to different behaviors on different carbon sources and overall slower growth and glycolytic flux for the humanized strains versus native yeast.

Three glycolytic yeast enzymes have secondary “moonlighting” roles beyond their function in glycolysis. Hexokinases are involved in glucose repression of genes such as invertase (SUC2), and, indeed, it appears that human hexokinases can also at least partially complement this secondary function. Yeast aldolase (FBA1) plays a secondary role in vacuolar function that is required for growth at alkaline pH.  This function is likewise complemented by the human orthologs, where the humanized strains can grow at pH 7.5. The third moonlighter is enolase, where yeast ENO2 is required for mitochondrial import of tRNALys, thereby allowing growth at higher temperatures and on non-fermentable carbon sources. All three human enolases can at least partially complement this growth defect, and thus appear to have the same secondary function.

Despite the high conservation of functions, the humanized yeast strains have a slow growth phenotype. The authors used this phenotype to employ adaptive laboratory evolution to see which genomic changes restore growth. Interestingly, the mutations that restored growth were mostly not in the glycolytic enzymes themselves, but in associated factors that regulate enzyme abundance and activity. The identity of these regulators and the potential for targeting them in human muscle have already rewarded the successful transfer of skeletal muscle glycolysis into yeast.

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, yeast model for glycolysis, glycolysis, humanizing yeast, muscle glycolysis

Link Between Aging and Iron

August 25, 2022

Perturbations in iron homeostasis affect aging, but how this happens has remained a bit of a black box. A new study by Patnaik et al. in Cell Reports illuminates this box by looking more closely at the transcription factors that are first to respond when iron becomes limiting.

Key among these are Atf1p and Atf2p, which activate the full suite of iron-mobilization genes, among which is TIS11/CTH2, which encodes an RNA-binding protein that targets specific messages for decay.

The targeted messages flagged for decay encode mitochondrial proteins, as these use iron but are not the most essential in the set. The most essential Fe-requiring enzymes are those involved in DNA synthesis and repair, such that slowing/shutting mitochondrial function is a response to iron deficiency. Intriguingly, mitochondrial function also happens to decline with age.

From Patnaik et al., 2022

To find the specific mechanisms linking iron with aging, the authors used an unbiased analysis of genes involved in iron homeostasis to see which showed connection with aging. The strain with a tis11Δ mutation lived longer than any others, with a lifespan extended by 51.1%. In a broader sense, they found that genes involved in different aspects of response to iron deficiency also had different effects on fitness and aging.

From Patnaik et al., 2022

Delving more deeply into the role of Tis11p/Cth2p in aging, the authors used RNA-seq and Ribo-seq to look at temporal changes in transcription versus translation in aging cells. They showed how, overall, aging leads to inhibition of translation—except for certain genes which are upregulated instead. Interestingly, most of the upregulated genes are in the Fe regulon that gets activated by the first responder Atf1p.

From Patnaik et al., 2022

While the expression of TIS11/CTH2 increases both with aging and with iron deficiency, the deletion of the gene extends lifespan. Thus, multiple lines of evidence suggest Tis11p/Cth2p is a negative regulator of longevity. The key connection appears to be mitochondrial translation, where the function of Tis11p/Cth2p to inhibit translation of mitochondrial transcripts for repressing non-essential Fe-requiring enzymes serves to simultaneously repress overall mitochondrial respiration, which speeds aging.

From Patnaik et al., 2022

As not all genes translationally upregulated in the tis11Δ mutant contained appropriate binding sites in the 3’ UTR, the authors looked further and found binding sequences for Puf3p, a protein known to bind and inhibit translation of mRNAs coding for mitochondrial ribosome proteins. Thus, Puf3p appears to be a critical partner for Tis11p/Cth2p in mediating downregulation of mitochondrial function. Further, they questioned the relationship with the Hap4p transcription factor, which regulates numerous components of the electron transport chain and whose overexpression extends lifespan. As the combination of a tis11Δ deletion with HAP4 overexpression had no additive effect in an epistasis experiment, they concluded that Tis11p-dependent repression acts through Hap4p.

The role of phosphorylation of Tis11p/Cth2p was examined by mutating N-terminal serine residues, which impairs degradation of the protein. Consistent with the converse result of extended lifespan in null mutants, the nondegradable version of the protein shortens lifespan.

Thus, the ease of the yeast model once more illuminates intricate connections between critical proteins, facilitating potential drug discovery around several new aging factors.

Categories: Research Spotlight

Tags: cell aging, aging, Saccharomyces cerevisiae, yeast model for aging, iron homeostasis

Sen1p Is the Traffic Cop for RNA Polymerase III

August 12, 2022

One way to imagine DNA is as a busy road with a lot of competing traffic. Say, a small village in southern Italy…where someone must mediate conflicts between competing vehicles to avoid disaster.

Illustration by Umberto Aiello, courtesy of the authors

It turns out that the “someone” in yeast cells is Sen1p. Two recent papers from associated groups describe the intriguing detail of how the Sen1p helicase plays this role for RNA polymerase III transcription. The paper by Aiello et al. in Molecular Cell shows how Sen1p regulates transcription-driven conflicts between the several machineries all engaged with DNA. In the related paper by Xie et al. in Science Advances, the authors show how the Sen1p helicase mediates “fail-safe” methods of transcription termination for RNA Pol III, thereby promoting efficiency and avoiding conflict with other pieces of machinery.

From Aiello et al., 2022

The key conflict preventing RNA Pol III from transcribing noncoding genes is with RNA Pol II, which is busy transcribing coding genes. Aiello et al. show how Sen1p has two strategies for mediating these conflicts, both of which involve interactions between Sen1p and the replisome. One involves temporary release of RNA Pol II from DNA while the other resolves genotoxic R-loops in nascent RNA. Both are critical for preventing genome instability.

from Xie et al., 2022

In the related paper by Xie et al., the authors focus on how termination of transcription of noncoding genes by RNA Pol III is achieved, and the role that Sen1p plays in termination. They show how Sen1p can interact with all three polymerases and also with the other two subunits (Nrd1p and Nab3p) of the NRD1 snoRNA termination (NNS) complex. More specifically, they show by mutation and co-immunoprecipitation that it is the N-terminal domain (NTD) of Sen1p that interacts with RNA Pol III and the replisome.

The authors use metagene analysis of RNA Pol II distribution at mRNA-coding genes to show how Sen1p can promote the release of RNA Pol II to resolve transcription-replication conflicts (TRCs). They further show how the association of Sen1p with the replisome is required for limiting TRCs at the ribosomal replication fork barrier, and how this action appears redundant with that of RNases H. The cooperation and redundancy in this role are key means to protect genome stability.

Not only is Sen1p required for termination of RNA Pol III transcription, but the authors show how this function is independent of the NNS complex. Unlike resolution of conflicts between RNA Pol II and RNA Pol III, the termination function of Sen1p does not require the replisome.

They asked the question of whether Sen1p acts via the primary termination site for RNA Pol III or, rather, a backup secondary termination that catches errors (i.e., when RNA Pol III reads through a weak termination site). Termination for RNA Pol III employs a tract of T nucleotides (T-tract) in the nontemplate strand and these T-tracts can be relatively weak or strong. When T-tracts prove insufficient to stop the polymerase, Sen1p plays a role by means of secondary structures in nascent RNAs, which act as auxiliary cis-acting elements. This backup method is termed the “fail-safe transcription termination pathway.” The RNA secondary structures are not absolutely required for RNAPIII termination, but can function as auxiliary elements that bypass weak or defective termination signals.

from Xie et al., 2022

Once more, it is the power of the yeast model that has allowed investigation to such exquisite molecular detail. That cells preserve genomic stability and avoid pile-ups amid so much traffic along DNA remains truly remarkable–even when we know more of how it works.

Categories: Research Spotlight

Tags: transcription, Saccharomyces cerevisiae, RNA polymerase II, transcription conflicts, RNA polymerase III, replisome

A Redox Switch that Remakes a Guide Protein into an Antioxidant

August 05, 2022

Cells are efficient machines, honed by selection. Recent studies are showing us how cells are more efficient than we realized, with proteins discovered to have multiple functions that can be surprisingly different under changes in environment. A new paper in Molecular Cell describes Get3p as one such protein. Under non-stress conditions, Get3p acts as an ATP-dependent guide for post-translational insertion of tail-anchored proteins into membrane. Under stress, however, Get3p morphs into an antioxidant “guard” protein that prevents toxic protein aggregation by functioning as a chaperone. What are the mechanics of such a switch?

The finding came about in something of a backward way, where Ulrich et al. set out to find a eukaryotic chaperone protein specifically activated during oxidative stress. They looked for similarity to Hsp33, a bacterial protein that protects against oxidative damage by activation of a zinc-coordinating, four-cysteine motif. Their search led them to Get3p, which shares little overall homology but has the same conserved CXC and CXXC motifs.

From Ulrich et al., 2022

Whereas the bacterial protein acts only in stress, yeast Get3p also has a non-stress role that differs from its role in stress response. Under non-stress conditions, it is the CXXC motif that plays the major role, where the two cysteines of the motif bind zinc, which aids the protein in dimerization. In the switch to the chaperone-active form, however, it is the nearby CXC motif that plays the critical role.

During oxidative stress, the switch to the chaperone form of the Get3p protein requires two things: (1) loss of bound ATP (with accompanying loss of ATPase activity, see below); and (2) oxidation of the free thiol groups (-SH) on residues C240 and C242 of the CXC motif. Once oxidized from thiol groups to sulfur groups, the two sulfurs of the CXC can form an intramolecular disulfide bond. Thus, in the presence of an ROS like H2O2, the new bond reconfigures the protein to become a non-ATP-requiring active chaperone. The CXC motif, then, acts as a “redox” switch.

From Ulrich et al., 2022

The role of ATP with respect to the redox switch is illustrated in the above schematic, where the presence of ATP makes the two thiol groups inaccessible to oxidation. When ATP is lost (due to the oxidative stress that reduces cellular ATP levels), the -SH groups can be oxidized to -S groups that bind to one another and partially unfold the protein, making it chaperone active.

The authors further show how the CXC motif is dispensable for the normal protein-targeting function of Get3p but absolutely required for the chaperone function. Thus, Get3p has two cysteine motifs that act in fully different ways, where CXXC is required for normal guide function while CXC is required for protection from oxidative stress.  

From Ulrich et al., 2022

Whereas the toxicity of reactive oxygen species is well established, the elucidation of a redox switch-triggered stress response in yeast provides an important clue to how eukaryotes detoxify these oxidants. It would not be surprising to find other cysteine-motif proteins acting as antioxidants in related processes that save cells from damage. As usual, yeast is an excellent model for understanding these intricate relationships.

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, oxidative stress, chaperones, redox switch

Insight into Evolution of Paralog Doublets

July 29, 2022

Sequencing of Saccharomyces cerevisiae in the 1990s revealed blocks of duplicated genes, suggesting an ancient whole-genome duplication. The original duplication has been postulated as a means to restore fertility to an interspecies hybrid of ancestral yeast parents; however, the question remains as to why so many paralog doublets have been retained through eons of evolution. Are these paralogs the residual clutter that has yet to be selected away, or has it represented an opportunity that created positive selection? Said another way, if every gene once had a twin, why have some been retained and others lost?

To dig further into understanding doublet evolution, a study by Purkanti and Thattai in a recent issue of Scientific Reports looks at the functional significance of these yeast paralog doublets from an evolutionary perspective. To ask whether retention of doublets across sub-clades of the yeast phylogenic tree might suggest selective advantage, they looked at which Gene Ontology biological processes showed the greatest enrichment of observed over expected for the 887 genes with conserved doublets. The greatest enrichment for over-represented doublets was in the GO process of endocytosis, with its parent term of vesicle-mediated transport also showing significant enrichment.   

Enriched biological processes; those related to vesicle traffic are in bold, from Purkanti and Thattai, 2022

The authors then assigned vesicle-traffic genes to function-specific and pathway-specific modules to ask about doublet retention. Among the modules, the greatest enrichment of doublets was observed for coat/adaptor genes and lipid control genes. Other modules, such as the endosomal sorting complex required for transport (ESCRT), had no doublets at all, i.e., the genes in this module have completely reverted to singletons.

Doublets per module, from Purkanti and Thattai, 2022

Intriguingly, the classes with singletons and the classes with doublets can be broadly grouped, where doublets tend to be retained in secretory and early endocytic pathways, whereas singletons largely act in retrograde Golgi traffic and late endocytic steps.

The authors separated all 360 ancestral vesicle traffic doublets into two groups: those that are retained as doublets in present-day species and those that are not. They looked at the nucleotide sequence identity as a proxy for evolutionary rate and found that retention of doublets is strongly associated with lower evolutionary rates. From this they conclude that doublet retention is under significant selection, as the purifying selection that lowers evolution rates is well correlated with functional significance.

While it may be mere coincidence that budding yeast happened to undergo ancestral genome duplication, it appears that this event has proven fortuitous for making S. cerevisiae even more useful as a model. As revealed in this study, the ease of manipulation and the vast knowledge base in yeast makes it possible to ask these deeply important questions about evolutionary impact on paralog doublets.  

Categories: Research Spotlight

Tags: paralogous genes, evolution, vesicle traffic, paralog doublets, Saccharomyces cerevisiae, whole genome duplication

Cln2p Moonlights in Cohesion

July 22, 2022

In an interesting new development, two different teams using two different sets of genetic interactions came to the same conclusion that Cln2p has a role in sister chromatid cohesion separate from its well-known role as a cyclin.

In a recent issue of the journal G3 (Bethesda), Buskirk and Skibbens report how deletion of the G1 cyclin CLN2 can rescue the temperature-sensitive growth defects of a strain lacking both ECO1 and RAD61, which are involved in regulation of sister chromatid cohesion. The authors show that neither CLN1 nor CLN3 deletion has the same effect, so the function is unique to CLN2. This result is especially interesting considering that CLN1 and CLN2 are paralogs resulting from whole genome duplication.

The authors show by genetic interaction how the role of Cln2p in sister chromatid cohesion is independent of its role in the G1/S cell cycle transition. Given that Cln2p activates Cdc28p within the CLN2-CDC28 kinase complex (Cln2-CDK) to trigger post-Start processes, the authors asked whether the whole complex has a role in cohesion. By looking closely at phenotypes of condensation, they conclude that this particular cyclin kinase complex plays a role in DNA hypercondensation, but that other CDKs interact with Eco1p at other points in the cell cycle.

Multifaceted and opposing roles for CDK regulation of Eco1 activity, from Buskirk and Skibbens, 2022

Using a similar but different approach, Choudhary et al. in a recent issue of mBio likewise looked for suppressors of a chromatid cohesion defect causing temperature-sensitive growth. In this case, they use a double mutant lacking Pds5p, a protein associated with the cohesin complex, and Elg1p, a protein involved in DNA replication. The elg1Δ mutation can suppress the pds5-1 temperature-sensitive allele but not the full deletion, such that the double mutant is inviable.

In a screen for mutants that rescued this lethality, 23 of 40 isolates carried mutations in the CLN2 gene. Deletion of the paralog CLN1 could not rescue the lethality of the pds5Δ elg1Δ double mutant, again suggesting these paralogs are not functionally redundant. Further investigation showed how cln2Δ deletion caused overexpression of MCD1, encoding the alpha-kleisin subunit of the cohesin complex. MCD1 has two promoter elements bound by the MBF transcription complex, and removal of these elements abrogated the overexpression. Thus, consistent with previous studies, the absence of CLN2 somehow caused overexpression of genes regulated by the MBF complex, in this case a critical cohesin subunit.

Model for the bypass of Pds5 function by elg1Δ cln2Δ, from Choudhary et al., 2022

We expect to hear more about the role of Cln2p in cohesion, for this secondary role is intriguing. It may be the case that other MBF-regulated genes are overexpressed when CLN2 is absent, and this might give rise to other roles. Given the complexity of these multifunction proteins, the yeast model provides a strong platform for answering the next questions.

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, checkpoint proteins, mitotic cell cycle, sister chromatid cohesion, cell cycle regulation

Acetylation Regulates the Nuclear Pore Complex

July 15, 2022

Several recent studies have done an excellent job characterizing the architecture of the yeast nuclear pore complex (NPC). With so much new information, researchers are now able to ask probing questions about how NPCs mediate communication between the nucleus and the rest of the cell. Considering that signals perceived from the environment need to reach the transcriptional machinery in the nucleus, and that mRNA transcripts made in response to these signals need to get back out to get translated, the NPC has a lot of communicating to do. A study in a recent issue of the EMBO Journal by Gomar-Alba et al. makes strong strides toward understanding how this communication is accomplished.

On the nuclear side of the NPC resides a substructure called the nuclear basket that has previously been shown to play roles in regulating gene expression and mRNA export. The nuclear basket also interacts with lysine acetyltransferases (KATs) and deacetylases (KDACs) that are best known for modulating transcription via reversible acetylation of histones in chromatin. These enzymes, however, can also act on non-histone proteins and have been linked to numerous cell processes, including DNA damage repair, cell division, and signal transduction.

From Gomar-Alba et al, 2022

Promotion of mRNA export is another function linked to acetylation, specifically by the NuA4 histone acetyltransferase complex, for which the catalytic subunit is Esa1p. Gomar-Alba et al. show in this recent study that Esa1p is the primary lysine acetyltransferase that promotes cell cycle entry—and also that it acetylates the nuclear pore protein Nup60p.

Acetylation of Nup60p promotes mRNA export, which in turn triggers fast entry into the Start phase of the cell cycle, thereby promoting cell division. Nup60p accomplishes this increased export by recruiting the TREX-2 transcription-export complex to the nuclear basket once Nup60p becomes acetylated. The deacetylated form of Nup60p has lower affinity for TREX-2 and thus mRNA export decreases. Deacetylation of Nup60p is performed by Hos3p, which acts in opposition to Esa1p in removing Esa1p-transferred acetyl residues.

Perhaps the most intriguing finding in this study is that Hos3p localizes primarily to daughter cells after cell division, causing displacement of the mRNA export complex and thus slowing G1/S phase transition. This action prevents premature division in the smaller daughter cells, as they require additional growth to meet the size control threshold for entry into a new cell cycle. Accomplishing this level of control with a single enzyme acting on a single nuclear pore protein is a simple, elegant solution.

As usual, studies in yeast make enormous impact on understanding cell division in other organisms.

Categories: Research Spotlight

Tags: cell cycle control, nuclear export, mRNA export, Saccharomyces cerevisiae, nuclear pore, acetylation

Pathway Between Copper and Disease Revealed in Yeast

July 08, 2022

Several lethal genetic disorders in humans are caused by mutations that cause symptoms of copper (Cu) deficiency, even in the presence of copper. These copper-deficiency disorders are fatal and include Menkes disease, Friedreich’s ataxia, and neurological and cardiac defects in infants due to lack of copper supply to cytochrome c oxidase in mitochondria. Given that no treatments are currently available for these terrible disorders, researchers have been interested in drugs that might improve copper bioavailability. The copper-binding oncological drug elesclomol (ES) has been identified as a candidate.

In a recent report by Garza et al. in the Journal of Biological Chemistry, the authors use the facility of yeast genetics to ask detailed questions about how ES affects metal homeostasis in yeast cells. It was previously established that perturbation in the levels of one metal tends to cause perturbations in supply of other metals, and thus they asked directed questions about both copper and iron (Fe). Deficiency in copper can cause linked deficiencies in bioavailable iron, and both are critically important for metal-dependent enzymes in mitochondria.

The crux of the team’s findings was that supplementing copper-deficient yeast cells with Cu-bound ES (ES-Cu) not only increased Cu levels, but nearly doubled Fe levels in mitochondria. They were able to show that ES transports copper by an alternate route that bypasses the major yeast copper importer (Ctr1p). While this is an intriguing and perhaps encouraging result, perturbations in metals have so much potential to be toxic that it remains critically important to understand the relationships between the components.

From Garza et al., 2022

The authors found that application of preformed ES-Cu complex is more efficient than ES at transporting Cu, and that transport of Cu across the plasma membrane by the drug occurs by passive transfusion, not active transport. Further, they made the critical discovery that copper delivered by ES goes first to the Golgi lumen, not directly to mitochondria as the authors had expected based on studies in other models. At the Golgi, the Cu is made available to the copper-transporting ATPase Ccc2p, which in turn assists in metalating Fet3p, a multicopper oxidase that oxidizes ferrous (Fe2+) to ferric iron (Fe3+). Once activated with copper in the Golgi, Fet3p-Cu is transported to the plasma membrane, where it oxidizes iron to Fe3+, the form that can be taken up by the iron transporter Ftr1p. This increased transport of iron leads to increased bioavailability in mitochondria. Thus, the link between copper and iron by means of an alternative copper transporter becomes more clear.

Interestingly, copper and iron metabolism are linked in both humans and yeast. The yeast protein Fet3p has two homologs in humans (ceruloplasmin and hephaestin), both of which are multicopper oxidase proteins critical for normal iron metabolism. From these powerful studies in yeast, it appears that disorders of Cu metabolism cause defects in Fe metabolism due to disrupted metalation of these cuproenzymes within the Golgi. Indeed, this is an intriguing finding that opens avenues for possible therapies, and it would be hard to imagine making this connection without the use of the yeast model.

Categories: Research Spotlight

Tags: copper transport, iron, cytochrome c oxidase, mitochondria, Saccharomyces cerevisiae, copper, iron transport, yeast model for human disease

Snf5p Senses pH to Combat Starvation

July 01, 2022

Yeast are keenly sensitive to internal pH. Several membrane proteins pump H+ ions out of the cell to keep the internal pH near neutral.  When carbon becomes scarce, however, it is essential for survival that these pumps get inactivated so the internal space is rapidly acidified. This acidification is postulated to conserve energy and trigger a number of subsequent pathways to combat starvation. Key among these adaptive responses is the derepression of the glucose-repressed genes. The well-studied SWI/SNF complex has been established as a key mediator for this, but the details of how the transcriptional boost is effected have not been known.

A recent study in eLife by Gutierrez et al. has shown the pivotal subunit of the SWI/SNF complex to be Snf5p, which performs a regulatory role by sensing pH. But how would a protein sense pH?

from Gutierrez et al., 2022

In studying the sequence and structure of the eleven subunits of the SWI/SNF complex, the authors noted that ten of the eleven subunits had large intrinsically disordered regions and that four of the eleven contained glutamine-rich low-complexity sequences (QLCs) that contain multiple histidine residues. QLCs were previously identified as important for binding transcription factors.

In looking for a link between pH and activation, the authors postulated that the histidine residues might be important because the histidine sidechain has an intrinsic pKa of 6.9, and thus might change conformation when pH drops.

Detailed comparative analysis of QLCs from yeast and other organisms led the authors to conclude that, in yeast, the histidines are salient features of QLCs that have been evolutionarily conserved. Given this, they noted that the N terminus of Snf5p has one of the largest QLCs in the whole yeast proteome and is in the top three for number of histidines.

from Gutierrez et al., 2022

Naturally, given the tools of the yeast model, the next step was to mutate the protein, for which they compared a full deletion against an N-terminal deletion of the QLC and a targeted allele with four histidines within the QLC mutated to alanine.

from Gutierrez et al., 2022

They found that total loss of the gene was phenotypically distinct from either of the QLC-targeted mutants. Total absence of the protein caused disruption of the SWI/SNF architecture, while QLC-mutants maintained an intact complex but showed disruptions in transcriptional reprogramming in response to starvation, as specifically measured by derepression of the ADH2 gene.

from Gutierrez et al., 2022

By a subsequent series of elegant biochemical experiments—conducted both in vivo and in vitro—the authors show with great precision how the Snf5p QLC specifically senses pH to trigger widespread reprogramming of genes that will help yeast metabolize non-preferred carbon sources. Even more specifically, they show how acidification leads to protonation of the histidines in the QLC, causing that region of the protein to expand and change conformation, thereby affecting the binding properties of the whole SWI/SNF complex.

from Gutierrez et al., 2022

The ability to do these experiments and develop a model of how the cell accomplishes delicate regulation once more astounds us with the awesome power of yeast genetics.

Categories: Research Spotlight

Tags: glucose-repressed genes, signal transduction, glucose metabolism, glucose starvation, pH sensing, Saccharomyces cerevisiae, glucose repression

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