Getting Folded Proteins Through the Doors of the Chloroplast
By: Tegan Armarego-Marriott
Tags: Biology, Science, Plants, Chloroplast, Scicomm

a.k.a Fold me twice, shame on me.

By Tegan Armarego-Marriott

If you’ve ever tried to buy second hand Ikea furniture- something big that’s already been assembled- you’ll be familiar with what I like to call ‘the door dilemma’.

It’s the question of how much you have to dissemble that new-to-you Billy bookcase, in order to get it through your front door. Or, perhaps more honestly, the question of whether your front doorway is wide enough to allow for your maximum laziness.

These kind of spatial guessing games are not unique to 20-somethings looking to furnish their apartment on the cheap, but in fact crop up throughout the natural world, including in the plant kingdom. And recent work from Iniyan Ganesan and colleagues suggests that when plants are faced with the’ door dilemma’, they, or at least their chloroplasts, might be just as lazy as us!

Plants, like many organisms, are all about specialization. They have specialized organs (roots, flowers, stems, and leaves), specialized cells (phloem to transport sugars, guard cells to open and close leaf air holes), and specialized compartments within the cells. These compartments, or organelles, include the ‘powerhouse of the cell’, a.k.a the mitochondria, the brain-like nucleus, and of course, the chloroplast.

In order to function in unique ways, different organelles must be separated from each other. For chloroplasts, it’s about keeping their inner environment right for the reactions of photosynthesis, but also keeping all of the nasties that come from those reactions (like reactive oxygen species) away from the rest of the cells. So the chloroplast, like other organelles, is separated from the cellular cytoplasm by a physical barrier- a lipid membrane.

Of course, in order for parts of the cell to work together, the different compartments can’t be completely closed off: they have to be able to exchange messages and goods. So the perimeter fence needs a door. Something that isn’t so big that everything falls out, but that’s big enough to let the right things in.

The ‘right things’ that need to get in through the chloroplast doorways (scientifically known as translocons) are proteins. Many of them. Chloroplasts are fairly useless on their own, and need to import literally thousands of proteins from the cytoplasm in order to function.

But, just like a Billy bookcase, proteins are huge 3-D beasts in their natural form. Which either means that chloroplasts have a whole lot of work ahead of them, in first in disassembling the protein into its 2-D chain-like form to get it through the translocons, and then reassembling the protein once it‘s inside. Or, it could just be that that chloroplasts have pretty big translocons.

To find out which is true, Ganesan and colleagues took advantage of a protein known as dihydrofolate reductase, or DHFR. DHFR has long been a favourite model protein for scientists, because it has a known size when folded, and, once you add its chemical partner methotrexate (MTX), it’s stays put in this folded form.

Previous experiments have shown if you add the DHFR-MTX complex to chloroplasts and wait a bit, you’ll find DHFR-MTX inside the organelle. This would seem to suggest that the whole complex fits through the translocon… but scientists questioned the result, wondering if perhaps the chloroplast was simply really good at ripping the DHFR-MTX apart.

Complicating this situation was the fact that MTX could readily diffuse through the chloroplast membranes, effectively avoiding the chloroplast doorways. So maybe the two were separated at the door, DHFR went through the translocons in its 2D form while MTX slipped through the membranes, and they met up again inside.

In order to avoid this complication, Ganasen and colleagues used a new chemical, FMTX: a version of MTX linked to a fluorescent dye. FMTX binds to the DHFR protein in the same way MTX does but has two advantages. Firstly, it fluoresces, or glows, and the intensity of the glow changes depending of whether or not it is bound to DHRF. This fluorescences allows quantification of the DHFR-FMTX complex. And secondly, unlike MTX, it’s not so good at sneaking through the membrane.

The DHFR-FMTX complex could enter the chloroplast by two methods. Firstly, the DHFR-FTMX protein could enter through the outer and inner chloroplast membrane translocons in its folded state (left image). Alternatively, the protein (DHFR) could be unfolded and separated from FMTX (right image), with both components entering the chloroplast independently. It should be noted that FMTX, unlike MTX, can not easily or rapidly move through the chloroplast membranes. Image adated from Ganesan et al., 2018.

By measuring the fluorescent signals coming from inside the chloroplast under different experimental conditions, the authors could show that FMTX import occurs fastest when DHFR is also being imported. And that inhibiting the import of DHFR results in proportional decreases in FMTX import.

Together, these results strongly suggest that DHFR and FMTX travel into the chloroplast together, and use the same doors to do so. This in turn, suggests that the chloroplast translocons themselves must be big enough to fit DHFR through in its folded form. Ganasen and colleagues supported this theory by showing that they could also get other DHFR-sized particles into the chloroplast.

What does all this mean? Well, the size of the chloroplast membrane translocons are much bigger than expected; bigger, for example, than those seen in the outer and inner membranes of the mitochondria, and bigger than certain known bacterial translocons. The authors even speculated that the size of the translocon pores may be flexible, allowing them to expand to accommodate certain large proteins. Or that the translocons themselves may exist in different forms, with different maximal sizes, around the chloroplast membrane.

On one hand this avoids this suggests that chloroplasts may have just opted for the lazy option when faced with the door dilemma, avoiding the disassembly routine for small proteins. But there are still proteins much larger than DHFR that have to get into the chloroplast, and therefore probably still have to be unfolded prior to import. And of course, a big door leads to big problems- all kinds of things can get in and out, which means you have to have a pretty good ‘doorman’. So despite the win for laziness, it’s hard to call this an out-and-out victory.

Which is kind of a thing in nature: everything in evolution is a trade-off!

To read more about the chloroplast translocon pore size, check out the original article:

Ganesan. I, Shi. L-X, Labs. M, Theg, S. M. (2018). Evaluating the Functional Pore Size of Chloroplast TOC and TIC Protein Translocons: Import of Folded Proteins. Plant Cell 30: 2161–2173; DOI: https://doi.org/10.1105/tpc.18.00427