Saturday, October 24, 2015

With a Little Help From My Chaperones

Ribosomes need hundreds of helper proteins during construction.

One of the premier machines of molecular biology is the ribosome. It weighs in at about 3 million daltons, or hydrogen atom-equivalents, and has a huge catalytic core of RNA surrounded by 79 proteins. Due to its ancient origin, mixed composition, and large size, it is also very complicated to produce, yet needs to be made in prodigious amounts. Its manufacture begins in its own organelle, the nucleosome, which is a small compartment within the nucleus where the many copies of genomic DNA that encode ribosomal RNAs get transcribed. Countless events happen thereafter, chemically modifying the RNA, adding proteins, chemically modifying them with various phosphate, acetyl, and methyl groups, and transporting the nascent ribosomal halves out of the nucleus to the cytoplasm. One irony is that the proteins added to the ribosome are all synthesized (by pre-existing ribosomes, naturally) in the cytoplasm and thus have to be transported into the nucleus individually before being re-exported as part of the assembled ribosome halves.

While most proteins and RNAs fold themselves and assemble naturally, based solely on their sequences / composition, the bigger they are and the bigger the complexes they participate in, the more help they tend to need from special proteins called chaperones. The ribosomal RNA uses 76 helper snoRNAs to get itself folded and modified correctly. For assisting the folding of proteins, there are two classes of helpers, general chaperones which help proteins fold by exposing them alternately to hydrophobic and hydrophilic surfaces, and specific chaperones that bind to one or a few target proteins, typically right as they come off the ribosome production line, to prevent them from aggregating with the wrong crowd, and to transport them to the right place for assembly. Assistance for ribosomal RNA folding may have been the original function of some ribosomal proteins which are now essential for function and permanent parts of the mature structure. But now the ribosomal proteins themselves need chaperones, to the tune of about 200, for proper assembly.

A recent paper discussed an example of a specific ribosomal chaperone, Acl4, which ferries the ribosomal protein Rpl4 to its mark. Rpl4 is an average-sized protein, about 50,000 daltons, but its structure is remarkably splayed-out, rather than compact. When assembled, part of its structure reaches into the exit tunnel of the ribosome, where newly synthesized protein chains come out, and seems to help them stay in the straight and narrow, especially hydrophobic segments that would be tempted to stick to themselves or other proteins, clogging the tunnel.
Structure of Rpl4. The blue part sticks into the protein chain exit tunnel, and is evolutionarily conserved, while the red part reads over the surface of the ribosome, touching several other ribosomal proteins, with unknown function.

Position of Rpl4 (red) on the ribosomal large subunit. PTC labels the peptide transferase center, or synthetic core of the ribosome, and the emerging amino acids chained into a protein are black circles. The hydrophobic knee of the nascent protein tunnel is where the key segment of Rpl4 (4) has a role, along with some other ribosomal proteins (17, 39).

But this ability to manage hydrophobic protein segments implies that Rpl4 is itself, in that region, hydrophobic, and thus prone to aggegation. This is in addition to the rest of the structure, which reaches across several other proteins on the ribosomal surface, in snake-like fashion. While researchers know that this latter structure is essential, they do not know yet what it does. This intriguing protein clearly needs help in assembly. The researchers hypothesized such a chaperone helper, and went out to find it using a tagged version of RPL4 with which they could easily co-purify whatever stuck to it, including several of its ribosomal protein colleagues. But there was one more protein, called Acl4. Unfortunately, the researchers didn't come up with this name themselves, but were scooped by others who published similar data only a few months before. So it goes.

Using a series of engineered deletions of the Rpl4 protein, the researchers show that Acl4 binds over the key hydrophobic area of Rpl4, as one would expect. They additionally show that Acl4 binds to Rpl4 even before it is fully synthesized, also as one would expect for a specialized protein chaperone. In yeast cells, neither protein is actually essential. Strains with either or both genes deleted still live, though grow very slowly. They would never survive in the wild.

Knowing the nuts and bolts of how our biological molecules operate, particularly the extraordinary lengths evolution has gone to fix and fine-tune systems that must have been functional enough in their much simpler, early incarnations, fosters an appreciation of the messiness of the molecular world. Sometimes huge size and complexity is a product of endless jury-rigging, not of exquisite design.

Drama of ribosomal synthesis, for a few actors. Rpl4 (green) is synthesized in the cytoplasm, and captured by Acl4 (purple). Then both transport to the nucleus, where they dock to the assembling ribosome, which is then in due time transported back out to its final destination. Acl4 cycles back by itself for another load. A minor pathway also exists in the absence of enough Acl4, where a generic nuclear transport conductor, importin, can bring Rpl4 into the nucleus, since Rpl4 contains the necessary targeting signals in its sequence.

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