New & Noteworthy

New Sequence, Chromosome, and Contig pages

August 25, 2014

New Sequence pages are now available in SGD for virtually every yeast gene (e.g., HMRA1 Sequence page), and include genomic sequence annotations for the Reference Strain S288C, as well as several Alternative Reference Genomes from strains such as CEN.PK, RM11-1a, Sigma1278b, and W303 (more Alternative References coming soon). Each page includes an Overview section containing descriptive information, maps depicting genomic context in Reference Strain S288C (as shown below) and Alternative Reference strains, as well as chromosomal and relative coordinates in S288C.

The sequence itself includes display options for genomic DNA, coding DNA, or translated protein.

Also available on each Sequence page are links to redesigned S288C Chromosome pages, links to new Contig pages for Alternative Reference Genomes, and a Downloads menu for easy access to DNA sequences of several other industrial strains and environmental isolates. The new Sequence, Chromosome, and Contig pages make use of many of the features you enjoy on other new or redesigned pages at SGD, including graphical display of data, sortable tables, and responsive visualizations. The Sequence pages also provide seamless access to other tools at SGD such as BLAST and Web Primer. Please explore these new pages, accessible via the Sequence tab on your favorite Locus Summary page, and send us your feedback.

Special Delivery for Cytotoxic Proteins

August 21, 2014

Like the USPS delivering a letter, yeast Cue5p & human Tollip recognize the ubiquitin “stamp” on cytotoxic proteins and present them to the “addressee” Atg8p. Image from Wikimedia Commons

Say you want to send a letter to your friend on the other side of the country. First off you’ll need to put the right address and postage on the envelope. Then you’ll need the U.S. Postal Service (USPS) to take your letter and deliver it to the right person. The stamp tells the USPS to deliver the letter, and the address indicates where it should be delivered (unimpeded by snow nor rain nor heat nor gloom of night, of course!).

It turns out something similar happens in human cells with aggregated proteins. Aggregated proteins are “stamped” by attachment of the small protein ubiquitin and “addressed” to the Atg8 protein. Atg8p triggers the aggregated proteins’ incorporation into autophagosomes for eventual degradation in the lysosome.

And just as it can be devastating if your mail doesn’t get to where it needs to go, so too can it be devastating for these aggregates to accumulate instead of being properly delivered. A buildup of these aggregates is a big factor in Alzheimer’s and Huntington’s diseases.

Enter the cellular USPS. Just as is the case for a prepared letter, the human cell has a service that delivers the ubiquinated proteins to the autophagosome, in the form of the protein adaptor p62 (SQSTM1) and its relative, NBR1.

These adaptor proteins can act as a postal service because they recognize both the aggregated proteins’ stamp (ubiquitin) and their addressee (Atg8p). Specifically, they each possess an ubiquitin-conjugate binding domain (UBA) and an Atg8-interacting motif (AIM). The protein p62 in particular has been shown to associate with protein aggregates linked to neurodegenerative diseases like Huntington’s disease.

In a new paper published in Cell, Lu et al. asked whether there is a link between the ubiquitin and autophagy systems in yeast. If so, yeast might provide some clues about diseases like Huntington’s. Proteins stamped with ubiquitin are known to be addressed to the proteasome for degradation in yeast, but no link between ubiquitination and autophagy had previously been seen, even though many central components of autophagy were actually first described in yeast.

Indeed, the authors showed that cells specifically deficient in the autophagy pathway (atg8∆, atg1∆, or atg7∆), accumulated ubiquitin conjugates under autophagy-inducing conditions. This suggests that the ubiquitin and autophagy pathways are connected in yeast, as is the case for humans.

Next, the researchers looked to see if there is an adaptor in yeast analogous to p62 in humans. When they pulled down proteins that bind yeast Atg8p under starvation conditions, they found ubiquitin conjugates and, using mass spectrometry, further identified peptides from a few other proteins – one of which was Cue5p.

Could Cue5p, like p62 in humans, be the postal service that recognizes both stamped ubiquitin conjugates and the addressee Atg8p in yeast? Strikingly, Cue5p had both a CUE domain that binds ubiquitin and an Atg8p-interacting motif (AIM). The authors confirmed in vivo that Cue5p binds ubiquitin conjugates and Atg8p using these domains, particularly under starvation conditions. They also showed that it acts specifically at the stage of ubiquitin-conjugate recognition and on aggregated proteins, without affecting the process of autophagy itself.

Given that Cue5p functions similarly to p62 and p62 is known to associate with protein aggregates involved in neurodegenerative disease, Lu et al. were quick to look for Cue5p substrates. Analyzing ubiquitin-conjugated proteins that accumulated in cue5 mutant cells, they identified 24 different proteins. Although these 24 Cue5p substrates had diverse functions, the common thread was that many had a tendency to aggregate under certain conditions such as high temperature.

Could Cue5p then actually facilitate removal of cytotoxic protein aggregates in neurodegenerative diseases? Indeed, the authors showed that CUE5 helped clear cytotoxic variants of the human huntingtin protein (Htt-96Q) when it was expressed in yeast, and that Htt-96Q is ubiquitinated in yeast.

These experiments started with an observation in human cells that prompted discovery of an analogous system and adaptor protein in yeast. Now the authors turned the tables and used yeast to look for new adaptor proteins in human cells. Using bioinformatics, they identified a human CUE-domain protein, Tollip, which, although different in its domain organization from Cue5p, contains 2 AIM motifs.

To make a long story (and a lot of work!) short, they showed that Tollip binds both human Atg8p and ubiquitin conjugates and clears cytotoxic variants of huntingtin in human cells. Expressed in yeast, it similarly binds ubiquitin conjugates and Atg8p and suppresses the hypersensitivity of cue5∆ cells to the variant huntingtin protein Htt-96Q. So Tollip is a newly defined adaptor protein and functional homolog of Cue5p!

Letter carriers of one sort or another have been around for as long as human civilization has existed, from homing pigeons to FedEx. Now we know that for even longer, cells from yeast to human have been using similar ways to recognize stamped proteins and deliver them to the right address. And once again, yeast has helped us understand the inner secrets of human cells.

Pinpointing Peroxisomes

August 14, 2014

The contents of the cell certainly move around, but they’re not quite as mobile as the blobs in this lava lamp. Image from Wikimedia Commons

One way to think about the cell is that organelles float around in it like those globs in a lava lamp.  This is obviously a simplification, but it’s also true that organelles aren’t locked into place.  As usual, the real picture lies somewhere in between these two extremes.

What we know about the architecture of the cell has mostly been discovered using classical cell biology and genetic techniques. But in a paper published in Molecular BioSystems, Cohen et al. uncovered some very interesting small details using a very large-scale approach.

The authors were interested in peroxisomes, where a lot of critical metabolic reactions happen (or fail to happen, in several human diseases). The researchers were able to see that peroxisomes not only interact with other organelles, but they contact the endoplasmic reticulum (ER) and mitochondria in a way that could be extremely important for cellular metabolism. And surprisingly, it was by combining a variety of different high-throughput techniques that Cohen and colleagues could uncover this fine structure.

The first step was to set up two reporter constructs to look for genes involved in two different peroxisomal processes.

One reporter was a red fluorescent protein, mCherry, modified to carry a peroxisomal targeting signal and show whether import into peroxisomes was normal. Another reporter, a peroxisomal membrane protein (Ant1p) tagged with green fluorescent protein (GFP), would show whether peroxisomal membranes were normal.

The reporters were crossed into mutant collections, creating one strain for each gene in the genome that had either a complete deletion (for nonessential genes) or a knock-down allele (for essential genes), plus both reporters. Now the researchers could systematically test for genes that, when mutated, affected one or both of these aspects of peroxisomal biogenesis.

To visualize the mutant phenotypes, they used a sophisticated technique termed “high-content screening.” This is an automated way to analyze micrographs that both pinpoints the intracellular location of a fluorescent reporter and measures its quantity. Screening the mutant collection in this way showed that 56 strains had altered distribution of the two different reporter proteins.  Some had a reduction in peroxisomal protein import (mCherry fluorescence), while some had fewer or no peroxisomes and some had peroxisomes that were smaller than normal (GFP fluorescence).

One result that caught the researchers’ eyes was that one of the strains with smaller peroxisomes had a mutation in the MDM10 gene. Mdm10p is part of the ERMES (ER-Mitochondria Encounter Structure) complex that tethers mitochondria to the ER, and this wasn’t previously known to have any connection with peroxisomes. Strains that were mutant in other ERMES subunits had the same phenotype, confirming that the complex has something to do with peroxisome structure.  Other results from the screens added weight to the idea of a three-way connection between peroxisomes, the ER, and mitochondria, and the authors went on to show that peroxisomes often sit at the ERMES complex where mitochondria contact the ER.

Next, to test whether mitochondria might have specific subdomains where peroxisomes interact, the authors used yet another large-scale screen. In the C-terminal GFP fusion library, where each yeast open reading frame is C-terminally tagged with GFP, 96 strains showed a punctate pattern of the fluorescent signal – meaning that the protein was concentrated in spots, rather than evenly distributed.  They labeled the mitochondria with a red fluorescent marker protein in these strains and, again using the high-content screening system, identified protein spots that co-localized with mitochondria. The most intense hit was for Pda1p, a subunit of the mitochondrial enzyme pyruvate dehydrogenase (PDH), and a similar result was obtained for another PDH subunit. So PDH isn’t distributed uniformly in the mitochondrion, but is instead concentrated in clusters.

Looking more closely using the various reporter constructs in their collections, the authors found that peroxisomes and the ERMES complex most often co-localized with those mitochondrial globs of PDH. It would make metabolic sense for peroxisomes to hang out near PDH on mitochondria because this could increase the local concentration of metabolites that they both use.

Intriguingly, Cohen et al. also found that mitochondria and peroxisomes co-localized in mammalian cells. Given that many diseases are linked to peroxisomal metabolism, this is an important avenue to investigate.

So while organelles don’t float around in the cell quite as fluidly as the globs in a lava lamp, the data generated from large-scale approaches boiled down to learning some very fine-grained detail about cellular architecture. We think that’s, like, groovy.

Shmoos Lost in Translation

August 7, 2014

Yeast cells don’t always shmoo…but when they do, they prefer eIF5A. Image courtesy of Gabriel Fox

To mate, the yeast Saccharomyces cerevisiae needs to shmoo — to generate a projection that reaches out to a nearby yeast of the opposite sex, until the yeast cell is shaped like the Al Capp cartoon character.  And to shmoo yeast needs, among other things, polyamines like spermidine.

Spermidine is important for one of the most interesting proteins in the world, the translation initiation and elongation factor eIF5A.  Not only is this protein pretty much conserved in just about every living thing, but it is also the only protein to have the unique amino acid hypusine.  And to make things even more fascinating, there are two other conserved proteins whose only job is to convert a single lysine residue of eIF5A into hypusine, using polyamines like spermidine.  Simply mind boggling.

In a new study in GENETICS, Li and coworkers provide compelling evidence that spermidine is important in yeast shmooing because of its involvement in the hypusinylation of eIF5A.   They also found that one reason eIF5A is so important in this process is that it is necessary for translating Bni1p, a formin needed to organize the actin cables of the shmoo.  Without these actin cables, the shmoo can’t form.

It looks like yeast needs eIF5A to translate Bni1p because of the long stretches of prolines found in this protein.  This suggests that like its bacterial ortholog EF-P, a key job for eIF5A is to help the cell deal with polyproline stretches in proteins.

To show this the researchers made a set of targeted mutations to check whether hypusinylation of eIF5A is necessary for shmooing.  When they knocked out LIA1, one of the enzymes that uses spermidine to convert lysine to hypusine, the resulting yeast failed to shmoo.  Since the only known target of the Lia1 protein is eIF5A, this suggests that hypusinylation of eIF5A is critical to its function in shmooing.

They also used temperature sensitive mutants of eIF5A to show that this gene (HYP2, also known as TIF51A) is involved in shmooing.  At the nonpermissive temperature, only 7.7% of yeast with the less severe mutant allele, tif51A-1, shmooed, while none of the yeast with the more severe mutation, tif51A-3, were able to shmoo.  These two results taken together establish the importance of eIF5A in shmooing.

Because eIF5A was known to be important for translating polyproline regions, the researchers looked for yeast proteins with such stretches, with the idea that their failure to be translated may be behind the need for eIF5A in shmooing.  They found 549 such proteins, and a comparison of their Gene Ontology (GO) annotations showed four overrepresented categories including “mating projection” (shmoo).  They focused on a protein from this group, Bni1p, because it was known to be involved in shmoo formation and it was one of only two proteins with ten or more prolines in a row. 

Bni1p is important for organizing the actin cables that are needed to make a shmoo.  Li and coworkers showed that the temperature sensitive mutants of eIF5A and bni1 mutants had similar phenotypes in terms of actin organization in the shmoo.

So the idea here is that yeast need eIF5A to shmoo because they need eIF5A to translate Bni1p, and Bni1p is needed to set up the actin framework of the shmoo.  In this hypothesis, it is the indirect action of eIF5A that prevents the shmooing. To test this hypothesis, the authors generated a bni1 mutant that lacked the polyproline regions. 

They compared the transcript levels of wild type BNI1 and the mutant lacking the polyproline stretches using RT-qPCR and found that the presence of eIF5A didn’t matter much.  The transcript levels of the mutant and wild type BNI1 were pretty much the same.

It was a different story for the protein levels.  Using Western blots Li and coworkers saw very little wild type Bni1p, but lots of the mutant protein.  The yeast cells struggled to translate wild type Bni1p but had no trouble with the mutant.  The easiest explanation is that eIF5A is needed to help the yeast translate polyproline regions of proteins, including Bni1p. 

Finally, to confirm the eIF5A and Bni1p connection, they showed that additional Bni1p could partially overcome the shmoo defect of the temperature sensitive mutants of eIF5A.  Since this suppression was only partial, and since the mutant phenotype of the eIF5A mutant is more severe than that of the bni1 mutant, there are probably other proteins involved in shmooing that require eIF5A for translation. Some likely candidates are those proteins containing polyproline stretches that are annotated to the GO term “mating projection”.

Although a connection between oddly-shaped yeast cells and human fertility and/or disease may not seem obvious, there might indeed be one. It turns out that eIF5A is so highly conserved  that human eIF5A works just fine when expressed in yeast, and mammalian formins, like Bni1p, are also proline-rich. Formins are necessary for polarized growth, which is a feature of both reproductive cell and cancer cell growth, and spermidine is required for fertilization.

Hard to believe that yeast channeling a cartoon character can teach us so much about the most fascinating of proteins, eIF5A.  And maybe even shed light on our own fertility. 

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