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Intelligent Design, the best explanation of Origins » Molecular biology of the cell » Signal Recognition Particle: An essential protein targeting machine

Signal Recognition Particle: An essential protein targeting machine

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Signal Recognition Particle: An essential protein targeting machine 1

Proteins destined for the cell membrane carry a marker sequence much in the way nuclear proteins carry an NLS. When the ribosome detects the marker sequence, it moves to the surface of the ER where it threads the protein through a pore as it is being synthesized. Ribosomes, located on the surface of the ER, can be clearly seen in electron micrographs; those areas are referred to as the rough ER. Once the protein is inside the ER, it is glycosylated by
several different enzymes that add the sugar molecules sequentially. This is analogous to a team of painters working on the same canvas. One painter might lay in the sky and ground, after which another paints the clouds and rocks. The glycosylating enzymes seem to follow a set of rules, because the same kinds of glycoproteins are produced over and over again, but the nature of those rules is still unclear. When the enzymes in the ER are finished, the glycoprotein is loaded into a transport vesicle (bubble) and sent to the Golgi complex. 6

All the difficulties and uncertainties of evolutionary reconstructions notwithstanding, parsimony analysis combined with less formal efforts on the reconstruction of the deep past of particular functional systems leave no serious doubts that LUCA already possessed at least several hundred genes.  In addition to the aforementioned “golden 100” genes involved in expression, this diverse gene complement consists of numerous metabolic enzymes, including  the subunits of the signal recognition particle (SRP)  3

Targeting of proteins to appropriate subcellular compartments is a crucial process in all living cells. Secretory and membrane proteins usually contain an amino-terminal signal peptide, which is recognized by the signal recognition particle (SRP) when nascent polypeptide chains emerge from the ribosome. 4

The Signal recognition particle (SRP) and its receptor comprise a universally conserved and essential cellular machinery that couples the synthesis of nascent proteins to their proper membrane localization. The past decade has witnessed an explosion in in-depth mechanistic investigations of this targeting machine at increasingly higher resolution. In this review, we summarize recent work that elucidates how the SRP and SRP receptor interact with the cargo protein and the target membrane, respectively, and how these interactions are coupled to a novel GTPase cycle in the SRP•SRP receptor complex to provide the driving force and enhance the fidelity of this fundamental cellular pathway

Proper localization of proteins to their correct cellular destinations is essential for sustaining the order and organization in all cells. Roughly 30% of the proteome is initially destined for the eukaryotic endoplasmic reticulum (ER), or the bacterial plasma membrane. Although the precise number of proteins remains to be determined, it is generally recognized that the majority of these proteins are delivered by the Signal Recognition Particle (SRP), a universally conserved protein targeting machine (14). 

The cotranslational SRP pathway minimizes the aggregation or misfolding of nascent proteins before they arrive at their cellular destination, and is therefore highly advantageous in the targeted delivery of membrane and secretory proteins. Despite the divergence of targeting machinery, the SRP pathway illustrates several key features that are general to almost all protein targeting processes: 

(i) the cellular destination of a protein is dictated by its ‘signal sequence’, which allows it to engage a specific targeting machinery; 
(ii) targeting factors cycle between the cytosol and membrane, acting catalytically to bring cargo proteins to translocation sites at the target membrane; and 
(iii) targeting requires the accurate coordination of multiple dynamic events including cargo loading/unloading, targeting complex assembly/disassembly, and the productive handover of cargo from the targeting to translocation machinery. 

Question : How could and would the protein find its way to the right destination without the signal sequence just right, right from the beginning ? 

Not surprisingly, such molecular choreography requires energy input, which is often harnessed by GTPase or ATPase modules in the targeting machinery. 

Cargo Recognition by the SRP

Timely recognition of signal sequences by the SRP is essential for proper initiation of cotranslational protein targeting. Signal sequences that engage the SRP are characterized, in general, by a core of 8–12 hydrophobic amino acids. 

The multiple conformational rearrangements in the SRP•FtsY GTPase complex provide a series of additional checkpoints to further reject the incorrect cargos. These include: 

(i) formation of the early intermediate, which is stabilized over 100-fold by the correct, but not incorrect cargos (Figure 3B, red arrow b); 
(ii) rearrangement of the early intermediate to the closed complex, which is ~10-fold faster with the correct than the incorrect cargos (Figure 3B, red arrow c); and 
(iii) GTP hydrolysis by the SRP•FtsY complex, which is delayed ~8-fold by the correct cargo to give the targeting complex a sufficient time window to identify the membrane translocon.

In contrast, GTP hydrolysis remains rapid with the incorrect cargo (t1/2 < 1s), which could abort the targeting of incorrect cargos (Figure 3B, arrow d). A mathematical simulation based on the kinetic and thermodynamic parameters of each step strongly suggest that all these fidelity checkpoints are required to reproduce the experimentally observed pattern of substrate selection by the SRP (40).

These results support a novel model in which the fidelity of protein targeting by the SRP is achieved through the cumulative effect of multiple checkpoints, by using a combination of mechanisms including 

cargo binding, induced SRP–SR assembly, and kinetic proofreading through GTP hydrolysis.  Additional discrimination could be provided by the SecYEG machinery, which further rejects the incorrect cargos (102).  Analogous principles have been demonstrated in the DNA and RNA polymerases (103104), the spliceosome (105), tRNA synthetases (106) and tRNA selection by the ribosome (107), and may represent a general principle for complex biological pathways that need to distinguish between the correct and incorrect substrates based on minor differences.

The crowded ribosome exit site

Accumulating data now indicate that the ribosome exit site is a crowded environment where multiple protein biogenesis factors interact. As a newly synthesized protein emerges from the ribosomal exit tunnel, it interacts with a host of cellular factors that facilitate its folding, localization, maturation, and quality control. These include molecular chaperones.

Many proteins need to enter the ER for modification with sugars this occurs at the same time that they are being synthesized by the ribosome translation begins with synthesis of a short signal peptide sequence a signal recognition particle a protein complex binds to the signal peptide while translation continues the SRP then binds to its receptor in the ER membrane anchoring the ribosome the ribosome binds its receptor and the signal peptide meets the protein translocator translation proceeds and the protein passes through the translocator the signal peptidase cleaves the signal peptide leaving the new protein molecule in the lumen of the endoplasmic reticulum

A non-mechanical example of irreducible complexity can be seen in the system that targets proteins for delivery to subcellular compartments. In order to find their way to the compartments where they are needed to perform specialized tasks, certain proteins contain a special amino acid sequence near the beginning called a 'signal sequence.'' As the proteins are being synthesized by ribosomes, a complex molecular assemblage called the signal recognition particle or SRP, binds to the signal sequence. This causes synthesis of the protein to halt temporarily. During the pause in protein synthesis the SRP is bound by the transmembrane SRP receptor, which causes protein synthesis to resume and which allows passage of the protein into the interior of the endoplasmic reticulum (ER). As the protein passes into the ER the signal sequence is cut off. For many proteins the ER is just a way station on their travels to their final destinations (Figure 10.3). 

Proteins which will end up in a lysosome are enzymatically ``tagged'' with a carbohydrate residue called mannose- 6-phosphate while still in the ER. An area of the ER membrane then begins to concentrate several proteins; one protein, clathrin, forms a sort of geodesic dome called a coated vesicle which buds off from the ER. In the dome there is also a receptor protein which binds to both the clathrin and to the mannose-6-phosphate group of the protein which is being transported. The coated vesicle then leaves the ER, travels through the cytoplasm, and binds to the lysosome through another specific receptor protein. Finally, in a maneuver involving several more proteins, the vesicle fuses with the lysosome and the protein arrives at its destination. During its travels our protein interacted with dozens of macromolecules to achieve one purpose: its arrival in the lysosome. 

Virtually all components of the transport system are necessary for the system to operate, and therefore the system is irreducible. And since all of the components of the system are comprised of single or several molecules, there
are no black boxes to invoke. The consequences of even a single gap in the transport chain can be seen in the hereditary defect known as Icell disease. It results from a deficiency of the enzyme that places the mannose-6-phosphate on proteins to be targeted to the lysosomes. I-cell disease is characterized by progressive retardation, skeletal deformities, and early death.

Transport by vesicles: when proteins are made on the rough endoplasmic reticulum (RER), they get loaded into the Golgi apparatus. They are then sorted, modified and packaged in vesicles made from the budding-off of the Golgi membrane and discharged.
Sorting signals directs the protein to the organelle. The signal is usually a stretch of amino acid sequence of about 15-60 amino acids long.
There are at least three principles that characterize all vesicles mediated transport within cells:

i. The formation of membrane vesicles from a larger membrane occurs through the assistance of a protein coat such as clathrin that engulfs the protein because an adapter protein such as adaptin binds both to the coat and to the cargo protein bringing both close together. 5
The adaptin traps the cargo protein by biding with it’s receptors. After assembly particles bind to the clathrin protein they assemble into a basket-like network on the cytosolic surface of the membrane to shape it into a vesicle. Their final budding-off requires a GTP-binding protein called dynamin.
ii. The process is facilitated by a number of GTP-binding proteins (ex; dynamin) that assemble a ring around the neck of a vesicle and through the hydrolysis of the phosphate group of GTP to GDP until the vesicle pinches off. In other words, GTP is one of the main sources of cellular energy for vesicle movement and fusion.
iii. After a transport vesicle buds-off from the membrane, it is actively transported by motor proteins that move along cytoskeleton fibers to its destination. The vesicle then fuses with a target membrane and unloads the cargo (protein). But in order to fuse a vesicle with the membrane of another compartment, they both require complementary proteins, which in this case is soluble N-ethylmalei mide-sensitive-factor attachment protein receptor or, ahem, SNARE present in the membrane – one for the vesicle (vesicular SNARE) and one for the target membrane (t-SNARE).

Each organelle and each type of transport vesicle is believed to carry a unique SNARE. Interactions between complementary SNAREs helps ensure that transport vesicles fuse only with the correct membrane.
Membrane fusing does not always follow immediately after docking (unloading of cargo), however it often waits for specific molecular signals.
COP stands for Coatamer proteins (COP) which is the second class of coat protein that mediates budding-off of vesicles from large membranes. It can be of two types I (anterograde; moving in a forward direction. Ex; from ER to Golgi) & II (retrograde; moving in a backward direction. Ex: from Golgi back to ER).

Thirty years ago, the components and pathway for SRP-dependent protein targeting were first elucidated in mammalian cells through in vitro reconstitutions in cell extracts (59). The identification of the SRP homologue in prokaryotes a decade later further highlighted the salient, universally conserved features of this pathway (1012). The biochemical accessibility of the bacterial SRP system has allowed for in-depth mechanistic investigations of this pathway, allowing us to understand its underlying molecular mechanism at unprecedented depth and resolution.

With the exception of the chloroplast SRP , SRP-mediated protein targeting is a strictly cotranslational process that begins when a nascent polypeptide destined for the ER or plasma membrane emerges from the ribosome (Fig. 1A).

Overview of the pathways and components of SRP. 
(A) Multiple pathways deliver newly synthesized proteins to the ER or plasma membrane, with the SRP pathway mediating the co-translational targeting of translating ribosomes (right) and post-translational targeting machineries mediating the targeting of proteins released from the ribosome. 
(B) Domain structures of the ribonucleoprotein core of SRP, comprised of the SRP54 (or Ffh) protein and the SRP RNA (left), and the bacterial SRP receptor (right).

The N-terminal signal sequence on the nascent polypeptide serves as the ‘signal’ that allows the ribosome•nascent chain complex (termed the RNC or cargo) to engage the SRP and, through interaction with the SRP receptor (SR), to be delivered to the vicinity of the Sec61p (or SecYEG in prokaryotes) translocon at the target membrane (Fig. 1A). There, the RNC is transferred to the Sec61p/SecYEG machinery, which either integrates the nascent polypeptide into the lipid bilayer or translocates it across the membrane to enter the secretory pathway. Meanwhile, SRP and SR dissociate from one another to mediate additional rounds of targeting (Fig. 1A).

2. Intelligent Design Creationism and Its Critics, page 251
3. Koonin, The logic of chance, page 331
6. The Cell, Panno, page 69

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The protein export pathway of eukaryotes is highly conserved. Protozoa, yeast, and mammalian cells use essentially the same mechanisms to translocate proteins into an ER, to glycosylate and sort them in the Golgi, and to export them across the plasma membrane by exocytosis. 1

Translocation Across the ER Membrane

Translocation of newly synthesized proteins across the ER membrane shows many similarities to translocation across the plasma membrane protein of bacteria. Proteins are prevented from folding in the cytoplasm. They are fed across the plasma membrane through a translocon, a proteinaceous pore, which has three subunits very similar to the bacterial proteins made by the secY, E, and G genes. By electron microscopy, these pores are rings about 8 to 10 nm in diameter, with a

central pore of 2 nm, sufficient to allow the passage of an extended, hydrated peptide of 1.1 nm in diameter. These pores can now be recognized. In yeast, proteins traverse pores in the ER by two different types of translocation mechanisms. One is an ATP-driven process that translocates proteins whose synthesis is complete. The other couples translation to the translocation process. In this transport mode, the ribosome is attached to the proteinaceous transport pore, the translocon, and

feeds the nascent train through the pore as it is being synthesized. Mammalian cells only have the cotranslation made of translocation. When translocation is co-translational, the nascent chain is recognized in the cytoplasm by a signal recognition particle, which stops further protein synthesis until the complex of ribosome, nascent chain, and signal recognition particle reaches the endoplasmic reticulum

Behe, Black Box, page 103

A new protein, freshly made in the cell, encounters many molecular machines. Some of the machines grab hold of the protein and send it along to the location it is destined to reach. In a little while I will follow a protein along one pathway from start to finish. 3 The space probe is shaped like a huge sphere. Inside the sphere are a number of smaller, self-contained spheres, each of which holds machinery for specialized tasks. In the biggest of the interior spheres—let's call it the «library»—are the blueprints for making all the machines in the space probe. These are not ordinary blueprints, however. They can be thought of as blueprints in braille—or perhaps as sheet music for a player piano—where physical indentations in the blueprint cause a master machine to make the machine for which the blueprint codes.

One fine day the space probe senses (by some mechanism we'll ignore) that it needs to make another battery crusher and to send the newly made machine to work in the garbage treatment room, where it will help in recycling old batteries. So the process to do that is set in motion: The blueprint for the battery crusher is photocopied in the library, and the blueprint copy floats over to a window in the library (remember; there's no gravity). On the edge of the blueprint are punch holes arranged in a special pattern, which exactly matches pegs on a scanner mechanism at the window. When the blueprint hooks onto the scanner; the window door opens like the shutter of a camera. The blueprint jiggles loose of the scanner and floats out of the library into the main area of the probe.
In the main area are many machines and machine parts; nuts, bolts, and wires float freely about. In this section reside many copies of what are called master machines, whose job it is to make other machines. They do this by reading the punch holes in a blueprint, grabbing nuts, bolts, and other parts that are floating by, and mechanically assembling the machine piece by piece.
The blueprint for the battery crusher, floating in the main area, quickly comes in contact with a master machine. Whirring, turning appendages on the master machine grab some nuts and bolts and start assembling the crusher. Before it assembles the body of the crusher, however, the master machine first makes a temporary «ornament» that marks the crusher as a machine that has to leave the main area.

In the main area is another machine, called a guide. The shape of the guide is exactly complementary to the shape of the ornament, and little magnets on the guide allow it to attach securely. As the guide snuggles up to the ornament it pushes down on the master machine's switch, causing the master machine to halt its construction of the crusher.
On the outside of one of the interior spheres (we'll call the sphere «processing room #1») is a receiving site that has a shape complementary to part of the guide and part of the ornament. When the guide, ornament, and attached parts bump into that shaped section, the master machine's switch is flipped back on, causing construction of the crusher to resume.
Right next to that shaped section is a window. When the ornament taps on the window (there's a lot of jostling going on), it activates a conveyor belt inside the processing room and the conveyor belt pulls the new battery crusher inside the processing room, leaving the master machine, blueprint, and guide on the outside.

As the crusher was being pulled through the window another machine removed the now-unnecessary ornament. Now, amazingly, constriction machines embedded in the flexible walls of processing room #1 cause a section of the wall to close in on and surround some of the machines, forming a new, free-floating subroom. The remainder of the wall that was left behind smoothly seals itself.
The subroom now floats a short distance through the main area before bumping into a second processing room. The subroom merges with the wall, and spills its contents into processing room #2. The battery crusher then passes through processing rooms #3 and #4 by mechanisms similar to those that took it from room #1 to room #2. It is in the processing rooms that machines receive the tags that direct them to their final destinations. An antenna is placed on the battery crusher and quickly trimmed down to make a very special configuration; the special shape of the trimmed antenna will tell other mechanisms to direct the crusher to the garbage treatment room.

In the wall of the last processing room are machines («haulers») with a shape complementary to that of the trimmed antenna of the battery crusher. The crusher sticks to the haulers, and that area of the wall begins to pinch off to form a subroom. Outside the subroom is another machine (the «delivery coder») with a shape that exactly complements the shape of a machine (the «port marker») sticking out of the garbage treatment room. The sub-room hooks up to the garbage treatment room through the two complementary machines. Another machine (the «gateway») then drifts by. The gateway has a shape that is complementary to a portion of the delivery coder and the port marker. When it sticks to them the gateway punches a small hole in the garbage treatment room, and the transit sphere merges with it, dumping its contents into the disposal. The battery crusher is able to begin its work.

Perhaps by this point in the book, the reader can easily see how the transport system that sent the battery crusher to its destination is irreducibly complex. If any of its numerous components is missing, then the crusher is not delivered to the garbage treatment room. Furthermore, the delicate balance of the system must be maintained; each of the many components that interlock must do so precisely and then disengage, and each must arrive and depart at the proper times. Any single error will cause the system to fail.

All of the fantastic machines in our space probe have direct counterparts in the cell. The space probe itself is the cell, the library is the nucleus, the blueprint is the DNA, the copy of the blueprint is RNA, the window of the library is the nuclear pore, the master machines are ribosomes, the main area is the cytoplasm, the ornament is the signal sequence, the battery crusher is a lysosomal hydrolase, the guide is the signal recognition particle (SRP), the receiving site is the SRP receptor, processing room 1 is the endoplasmic reticulum (ER), processing rooms 2 through 4 are the Golgi apparatus, the antenna is a complex carbohydrate, the sub-rooms are coatomer or clathrin-coated vesicles, and various proteins play the roles of the trimmer, hauler, delivery coder, port marker, and gateway. The garbage treatment room is the lysosome.

Let's quickly run through a description of how a protein that is synthesized in the cytoplasm eventually finds its way to the lysosome. This will take just one paragraph. Don't worry if you rapidly forget the names and procedures of cellular transport; the purpose is simply to give you a glimpse of the cell's complexity.

1. The encyclopedia of molecular biology

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