New & Noteworthy

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.

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight, Yeast and Human Disease

Tags: Saccharomyces cerevisiae, mitochondria, endoplasmic reticulum, peroxisomes

Anchors Aweigh for Peroxisomes

August 15, 2013

Suppose you had a fleet of rowboats that you wanted to split up evenly between two shores.  You probably wouldn’t just set them free and hope they drifted to the right places. A better way might be to run some ropes from the distant shore and pull half the rowboats across.  That way you’d be sure to get the distribution of boats that you wanted.

Cells anchor their peroxisomes as carefully as fishermen anchor their boats.

Dividing cells face a similar problem with their organelles.  When cells divide, they need to make sure mother and daughter receive the right number of organelles.  Otherwise one or both could die!

This is obviously too important to just leave to chance, which is why cells have devised ways to precisely control how many organelles end up in mother and daughter.  But not every organelle is divided in the same way.  In our boat analogy, some are pulled over with ropes, others with chains, some with winches and so on. 

We don’t know a lot about how some organelles are distributed between mother and daughter cells.  For example, the details have not been worked out for peroxisomes, the organelles that contain enzymes for beta-oxidation of fatty acids. Until now, that is…

In a recent study in The EMBO Journal, Knoblach and colleagues looked in great detail at how yeast cells distribute their peroxisomes. During budding, some peroxisomes stay tied up in the mother cell while others are transported into the bud and re-tied there.  

The authors found that the structure that ties up peroxisomes is like a rope with hooks at both ends. One hook attaches to the peroxisome, while the other hook attaches to the cortical endoplasmic reticulum (ER) near the cell wall. Surprisingly, the same protein, Pex3p, acts as the hook at both ends of the rope, connecting it to both ER and peroxisomes.

The authors already knew that some peroxisomes stayed anchored around the edges of the mother cell while others were “mobile” and moved to the daughter when yeast cells divided. They also knew that the protein Inp1p was important for anchoring peroxisomes. In the inp1 null mutant all the peroxisomes are mobile and end up in the bud, while overexpressing Inp1p causes all the peroxisomes to be anchored in the mother cell and stay there.

Knoblach and colleagues suspected that Inp1p might act as the rope that tethers peroxisomes. To test this, they fused Inp1p to a protein that sits in the mitochondrial outer membrane, Tom70p. Now peroxisomes in this strain were attached to mitochondria! This established that Inp1p is the tether.

Another major molecular player in this process is Pex3p. The pex3 null mutant phenotype looks a lot like the inp1 mutant phenotype: the mother cell loses all its peroxisomes and they end up in the bud. Pex3p is an integral membrane protein that is channeled through the ER on its way to the peroxisomal membrane – so it can be present in both places. The authors found that both the N terminus and C terminus of Inp1p bind to Pex3p. All this suggested that together, Inp1p and Pex3p might form a structure that links peroxisomes to the ER.

They were able to show that Inp1p and Pex3p interact directly both at the peroxisome and at the ER using a neat trick called bimolecular fluorescence complementation. This simply means that if the two halves of green fluorescent protein (GFP) are brought close to each other, they can fluoresce like the intact protein.  The basic idea is that they fused the first half of GFP with Inp1p and the second half with Pex3p and looked for green spots to turn up in the right place of the cell.  Of course this is easier said than done!

To pull this off, the authors had to first make two haploid strains of opposite mating types. The first had a pex3 mutation that caused all Pex3p to be stuck in the ER, anchoring all its peroxisomes there. It also carried a version of INP1 that was fused to half of GFP.

The second strain had a different pex3 mutation that set all peroxisomes adrift, and this mutant pex3 gene was fused to the other half of GFP. This strain also had an additional marker that made peroxisomes glow red.

When the cytoplasms of these two strains had a chance to mix after mating, the zygote had red peroxisomes with glowing green spots, showing that Inp1p-half GFP from the first strain was interacting with Pex3p-half GFP from the second strain.  Because the Inp1p-half GFP of the first strain was bound to ER-localized Pex3p, and the Pex3p-half GFP of the second strain was localized to peroxisomes, this result showed that Inp1p connects peroxisomes with the ER.

The authors studied the kinetics of this process in a lot more detail and even tracked the wanderings of individual peroxisomes. The model that comes from all this work is that at start of budding, some peroxisomes are bound to Inp1p and others aren’t. Those that aren’t bound to Inp1p move to the bud, and the others stay anchored to the mother cell’s ER. Meanwhile, during budding Pex3p passes through the ER and re-emerges at the ER membrane at the bud cortex. It can then bind Inp1p, which in turn binds to the Pex3p on the surface of the migrating peroxisomes to dock them in the bud.

Not only is this cool just from a basic biology perspective, but it may also help us deal with some human peroxisome biogenesis disorders.  For example, Zellweger syndrome and infantile Refsum disease are associated with specific mutations in the human ortholog of PEX3. Once again, little S. cerevisiae is helping us navigate the inner workings of eukaryotic cells.  

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight, Yeast and Human Disease

Tags: yeast model for human disease, Saccharomyces cerevisiae, peroxisomes

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