Peroxisomes use a trendy way to import their proteins.
As has been discussed many times in this space, membranes are formidable barriers ... at the molecular level. Having a plasma membrane, and organelles enclosed within membranes, means needing to get all sorts of things across them, from the tiniest proton to truly enormous mega-complexes like ribosomes. Almost eight percent of the proteins encoded by the human genome are transporters, that concern themselves with getting molecules from one place to another, typically across membranes. A critical type of molecule to get into organelles is the proteins that belong there, to do their day-in, day-out jobs.
But proteins are large molecules. There are two ways to go about transporting them across membranes. One is to thread them across linearly, unfolding them in process, and letting them refold once they are across. This is how proteins get into the endoplasmic reticulum, where the long road to secretion generally starts. Ribosomes dock right up to the endoplasmic reticulum membrane and pump their nascent proteins across as they are being synthesized. Easy peasy.
However other organelles don't get this direct (i.e. cotranslational) method of protein import. They have to get already-made full-length proteins lugged across their membranes somehow. Mitochondria, for instance, are replete with hard-working proteins, virtually all of which are encoded in the nucleus and have to be brought in whole, usually through two separate membranes to get into the mitochondrial matrix. There are dedicated transporters, nicknamed the TOM/TIM complexes, that thread incoming proteins (which are detected by short "signal" sequences these proteins carry) through each membrane in turn, and sometimes use additional helpers to get the proteins plugged into the matrix membrane or other final destination. Still, this remains a protein threading process, (of the first transport type), and due to its need to unfold and the later refold every incoming protein, it involves chaperones which specialize in helping those proteins fold correctly afterwards.
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| Schematic of the nuclear pore. The wavy bits are protein tails that are F-G rich (phenylalanine-glycine) that are unstructured and form a gel throughout the pore, allowing like-minded F-G proteins through, which are the nuclear transport receptors. These receptors carry various cargo proteins in an out of the nucleus, without having to unfold them. "Nup" is short for nuclear pore protein; GLFG is short for glycine, leucine (another hydrophobic amino acid), phenylalanine, glycine. |
But there is another way to do it, which was discovered much more recently and is used principally by the nucleus. The nuclear pore had fascinated biologists for decades, but it was only in the early 2000's that this mechanism was revealed. And a recent paper found that peroxisomes also use this second method, which side-steps the need to thread incoming proteins through a pore, and risk all the problems of refolding. This method is to use a curiously constructed gel phase of (protein) matter that shares some properties with membranes, but has the additional property that specifically compatible proteins can melt right through it.
The secret lies in repetitive regions of protein sequence that carry, in the case of the nuclear pore, lots of F-G sequences. That is, phenylalanine-glycine repeated regions of proteins that form these transit gel structures, or pores. The phenylalanine is hydrophobic, the glycine is flexible, and the protein backbone is polar, though not charged. This adds up to a region that is a totally disordered mess and forms a gel that can keep out most larger molecules, like a membrane. But if encountered by another F-G-rich protein, this gel lets it right through, like a pat of butter through oil. It also tends to let small molecules through quite easily. The nuclear pore is quite permeable to the many chemicals needed for DNA replication, RNA production, etc.
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| Summary from current paper, making the case that peroxisomes use PEX13 to make something similiar to the nuclear pore, where targeted proteins can traverse easily piggybacked on carrier proteins, in this case PEX5. The yellow spaghetti is the F-G or Y-G protein tails that congregate in the pore to make up a novel (gel) phase of matter. This gel is uniquely permeable to proteins carrying the same F-G or Y-G on their outsides, as does PEX5. "NTR" is short for nuclear targeting receptor, to which nuclear-bound cargoes bind. |
Peroxisomes are sites for specialty chemistry, handling some relatively dangerous oxidation reactions including production of some lipids. They combines this with protective enzymes like catalase that quickly degrade the resulting reactive oxidative products. This suggests that the peroxisomal membrane would need to be pretty tight, but the authors state that the gel-style mechanism used here allows anything under 2,000 Daltons through, which certainly includes most chemicals. Probably the solution is that enough protective enzymes, at a high local concentration, are present that the leakage rate of bad chemicals is relatively low.
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| Experimenters purify large amounts of the Y-G protein segments from PEX13 and form macroscopic gels out of them. In the center is a control, where the Y residues have been mutated to serine (S). N+YG refers to the N-terminus of the PES13 protein plus the Y-G portion of the proteins, while Y-G alone has only the Y-G segment of the PEX13 protein. |
For its gel-containing pore, the peroxisome uses (on a protein called PEX13) tyrosine (Y) in place of phenylalanine, resulting in a disordered gel of Y-G repeats for its structure. Tyrosine is aromatic, (thus hydrophobic) like phenylalanine and tryptophan, and apparently provides enough distinctiveness that nucleus-bound proteins are not mistaken in their destination. The authors state that it provides a slightly denser packing, and by its composition should help prevent nuclear carriers from binding effectively. But it isn't just the Y-G composition that directs proteins, but a suite of other proteins around the peroxisomal and nuclear pores that, I would speculate, help attract their respective carrier proteins (called PEX5 in the case of peroxisomes) so that they know where to go.
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| Evolutionary conservation of the Y-G regions of PEX13, over a wide range of species. The semi-regular periodicity of the Y placements suggests that this protein forms alpha helixes with the Y chains exposed on one side, more or less, despite general lack of structure. |
The authors show some very nice experiments, such as making visible gels from purified / large amounts of these proteins, and then showing that these gels indeed block generic proteins, and allow the same protein if fused to PEX5 to come right through. The result shown below is strikingly absolute- without its peroxisome-specific helper, the protein GFP makes no headway into this gel material at all. But with that helper, it can diffuse 100 microns in half an hour. It is like making jello that you can magically pass your hand through, without breaking it up ... but only if you are wearing the magic glove.
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| Experimental demonstration of transport. Using macroscopic gel plugs like those shown above, the diffusion of green fluorescent protein (GFP) was assayed from a liquid (buffer) into the gel. By itself (center, bottom), GFP makes no headway at all. But when fused to the PEX5 protein, either in part or in whole, it diffuses quite rapidly into the Y-G gel. |
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