Protein targeting to peroxisomes and nuclei

Protein Targeting--Peroxisomes and Nuclei

PEROXISOMES:
Peroxisomes are different from mitochondria and chloroplasts in several ways: they have only a single outer membrane and a single internal space, and they contain no genetic information, so all their constituents are made in the cytosol and imported. However, like mitochondrial and chloroplast proteins, peroxisome proteins must be correctly targeted to the peroxisome and taken up through the membrane into the interior space, or else incorporated into the peroxisomal membrane. Not a lot is known about how these processes work, but what’s suspected is indicated in Fig.17.10 of Lodish et al. . Again we believe that there must be a specific signal sequence in peroxisomal proteins and specific receptors for this signal in the membrane of the peroxisome that somehow enables the protein to traverse the membrane. The signal sequence is probably three amino acids--serine-lysine-leucine--at the C-terminus of the protein (the last part syntheisized). At least part of the receptor/transport complex in the peroxisomal membrane is made of a pex14p protein. What's a little different about peroxisomes, is that the proteins are escorted to the peroxisome by a specific complex called the "peroxisome targeting sequence 1 receptor" (PTS1R). Apparently the PTS1R bind both to the SKL sequence in the perosixomal protein and to pex14p, serving as a kind of molecular matchmaker. There seems to be a separate escort protein, PTS2R, that recognizes a different, N-terminal peroxisome targeting sequence. What targets proteins to the peroxisomal membrane, and what supplies the energy for uptake isn’t known, nor have most of the components been identified.
However, several human diseases are caused by mutations that impair the functioning of peroxisomes; often these mutations affect targeting and uptake of peroxisomal proteins. One group of such diseases is called Zellweger’s syndrome, which is actually several kinds of peroxisomal storage diseases (that is, their peroxisomes fill up with undigested fatty acids, because the enzymes needed to degrade them are not present in the peroxisomes). Most Zellweger's patients have a mutation in the PTS proteins, but others have normal PTS proteins, suggesting that they have a mutation in other proteins involved in uptake of components of peroxisomes, and it's hoped that studying cells from these patients will help to identify other necessary components of peroxisomal uptake.
A second type of disease involving peroxisomes is adrenoleukodystrophy (featured in the film "Lorenzo's Oil"). These patients peroxisomes that lack long chain fatty acyl Coenzyme A synthesase (the first reaction in breakdown of fatty acids by beta-oxidation), so the peroxisomes become engorged with fatty acids that they can't metabolize. It's thought that the ALD mutation inactivates a transporter necessary for uptake of long-chain fatty acyl CoA synthase into the peroxisome.
NUCLEUS
The nucleus is the final organelle that I want to discuss. Although the nucleus contains the chromosomes whose DNA encodes most of the cell’s proteins, and though the DNA is copied into RNA in the nucleus, the nuclear proteins are made in the cytoplasm. The nucleus contains a large number of proteins, none of which is synthesized there. Can you give me some idea of the kinds of proteins that you'd expect to find in the nucleus? Answers could be: DNA replication enzymes, histones, RNA transcription and processing enzymes, gene regulatory proteins, and the like. Since ribosomes are assembled in the nucleus (nucleolus) ribosomal proteins are also present in nucleus.
These proteins are all made on ribosomes in the cytosol, so how do they get into the nucleus? The process for movement of proteins into (and out of) the nucleus seems to be substantially different from the others we just discussed, though there are some principles that it has in common with protein uptake by mitochondria, chloroplasts and peroxisomes. As you read in Lodish, the nucleus is surrounded by a double membrane. (Fig. 5-42b) The outer membrane is continuous with the ER, and is separated by a narrow gap from the inner membrane. However, the nucleus also contains unique structures called nuclear pores that are proteinaceous holes or pores that span both membranes. (Fig. 11-28 a & b) These pores are relatively nonselective and allow molecules up to about 5,000 MW pass in and out readily. (5,000 is a very small protein (50 aas) but is bigger than nucleotides, sugars, amino acids, etc.). Proteins between about 5000 and 60,000 Da enter progressively more slowly and anything bigger than that can't spontaneously pass through the pore. (RNA and DNA polymerase are proteins of >100,000 MW). For the most part, all the proteins that enter the nucleus do so in a native (completely folded up) state; thus chaperone proteins do not seem to be necessary. How do they get in? And how do ribosomes, or mRNA and its associated proteins get out of the nucleus?
For both nuclear import and nuclear export, it's thought that a group of specific exporter proteins is required to move large materials through the nuclear pores. These proteins include exportins, for export, importins, for import, and a small GTP-binding protein called Ran for movement in both directions. The movement of large materials through the nuclear pore uses up GTP and ATP--in other words, this is active transport.
What is currently thought is proteins that will enter the nucleus contain a "nuclear localization sequence" (NLS) and tthat there are two proteins in the cytosol, called "importins" a b , that bind to the NLS sequence, and then the complex of nuclear protein plus importin interacts with receptor proteins that are part of the nuclear pore. As a result the nuclear actively transports the protein into the nucleus. (Fig. 11-37). Once inside the nucleus, another protein, called Ran is necessary to dissociate the nuclear protein/importin complex. It displaces a importin by binding to b importin, thus releasing the cargo protein. The alpha importin and beta importin/RanGTP complex then exit the nucleus.
The fact that these proteins and others can also exit the nucleus means that there must also be an export mechanism for protein, mRNA, ribosomes, etc. Proteins like alpha and beta importin contain a poorly defined targeting signal called a Nuclear Export Sequence (NES) that allows them to exit nuclei through the nuclear pores. It's thought that they bind to a protein called exportin 1 plus Ran/GTP and the whole complex binds to and transits through the nuclear pores (Fig. 11-33). Once in the cytosol, the Ran is nudged by a protein called Ran GTPase-Activating Protein (Ran-GAP) to break down GTP to GDP. That changes its structure and it turns loose the exportin 1 and cargo protein. Ran and exportin 1 both contain NLS's, and so they move back into the nucleus to repeat the cycle, though Ran often associates with importins and cargo proteins before it reenters the nucleus, as we've already seen.
Ran is thus crucial to both nuclear import and export, and its function is dependent on whether it is bound to GTP or GDP. Two accessory proteins, RanGAP and a Ran nucleotide exchange factor, called RCC1 for some reason, are involved. RanGAP promotes breakdown of GTP to GDP by Ran, while RCC1 promotes exchange of GTP for bound GDP by Ran in the nucleus.
How can cell biologists identify where a protein is located in a cell? Ask for responses. There are several ways, all involving marking or tagging the protein in some way, and then finding it in the cell by microscopy. Sometimes one can use light microscopy, but because this does not have high resolution, it's more common to use electron microscopy. The principal is the same. For example, one can attach a fluorescent tag to the protein, inject it into the cell, and see what parts of the cell become fluorescent, or incubate the cell with fluorescent antibody to the protein and ditto.. If the protein stays in the cytoplasm, it will be clearly distinguishable from the nucleus and vice versa. To make a protein visible in the EM, you need to identify it with a label that reflects electrons, which is almost always a heavy metal atom. Such atoms are electron dense; they show up as dark places or spots in an electron micrograph. One that is commonly used is colloidal gold, little microspheres of gold of a certain size that one can coat with protein. Whereever the protein goes, the gold goes also, and one can readily see the gold particles as small black circles in electron microcrographs. A disadvantage of gold labelling is that you have to purify the protein that you're studying, mix it with the gold, then put it back into the cell.
What kind of experiment would allow you identify the nuclear signal sequence? Discuss. By using such experiment the NLS that was necessary and sufficient to target proteins to the nucleus was identified. One of the first proteins studied was from a virus (SV40), called T-antigen, that is normally localized to the nucleus, but there are mutant forms of the protein that aren't. This allowed identification of a very basic 7 amino acid sequence--PKKKRKV-that was necessary for nuclear uptake. When this sequence was attached to a cytosolic protein, pyruvate kinase, the PK was then localized to the nucleus (Fig. 11-35). Thus, these 7 amino acids have all the information necessary to direct a protein into the nucleus; they make up a Nuclear Localization Sequence (NLS). Not all NLS's are basic amino acids; some seem to comprise hydrophobic amino acids as well. Even less is known about NES's.
To recapitulate organelle biogenesis: Many cellular organelles that are surrounded by lipid/protein membranes contain proteins made on free ribosomes in the cytosol--including mitochondria, chloroplasts, peroxisomes and nucleus. The details of protein import into these organelles is different in each case, but all have some common principles. First, the proteins themselves contain some identification sequence--such as an NLS or mitchondrial import sequence--present only on proteins destined for a particular compartment. Second, the compartment contains in its outer membrane a complementary receptor protein or proteins that can bind to the signal sequence and get the protein to the appropriate location. Once this happens, some sort of transmembrane pore must assist the protein in passing through the bilayer--small pores in mitochondria, chloroplasts and peroxisomes and big ones in the nucleus. Finally, there are lots of accessory proteins required--different ones for different compartments, including things like the chaperone proteins, and the importins and exportins. While the general outlines of the process of organelle biogenesis are therefore becoming clearer, this is an area of intense study and progress, and it’s clear that our knowledge will change rapidly in the next few years.