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The SRP interacts with the SRP receptor


KEY TERMS:
  • The Alu domain comprises the parts of the 7S RNA of the SRP that are related to Alu RNA.
  • The S domain is the sequence of 7S RNA of the SRP that is not related to Alu RNA.
KEY CONCEPTS:
  • The SRP is a complex of 7S RNA with 6 proteins.
  • The bacterial equivalent to the SRP is a complex of 4.5S RNA with two proteins.
  • The SRP receptor is a dimer.
  • GTP hydrolysis releases the SRP from the SRP receptor after their interaction. 

The interaction between the SRP and the SRP receptor is the key event in eukaryotic translation in transferring a ribosome carrying a nascent protein to the membrane (for review see Keenan et al., 2001). An analogous interacting system exists in bacteria, although its role is more restricted.


The SRP is an 11S ribonucleoprotein complex, containing 6 proteins (total mass 240 kD) and a small (305 base, 100 kD) 7S RNA. Figure 8.24 shows that the 7S RNA provides the structural backbone of the particle; the individual proteins do not assemble in its absence (Walter and Blobel, 1981; Walter and Blobel, 1982).
The 7S RNA of the SRP particle is divided into two parts. The 100 bases at the 5 end and 45 bases at the 3 end are closely related to the sequence of Alu RNA, a common mammalian small RNA. They therefore define the Alu domain. The remaining part of the RNA comprises the S domain.
Different parts of the SRP structure depicted in Figure 8.24 have separate functions in protein targeting. SRP54 is the most important subunit. It is located at one end of the RNA structure, and is directly responsible for recognizing the substrate protein by binding to the signal sequence(Zopf et al., 1990). It also binds to the SRP receptor in conjunction with the SRP68-SRP72 dimer that is located at the central region of the RNA. The SRP9-SRP14 dimer is located at the other end of the molecule; it is responsible for elongation arrest (Siegel and Walter, 1988). 







The SRP is a flexible structure. In its unengaged form (not bound to signal sequence), it is quite extended, as can be seen from the crystal structure of Figure 8.24. Figure 8.25 shows that binding to a signal sequence triggers a change of conformation, and the protein bends at a hinge to allow the SRP54 end to contact the ribosome at the protein exit site, while the SRP19 swings around to contact the ribosome at the elongation factor binding site (Halic et al., 2004). This enables it to cause the elongation arrest that gives time for targeting to the translocation site on the membrane.
The SRP receptor is a dimer containing subunits SRα (72 kD) and SRβ (30 kD) (Tajima et al., 1986). The β subunit is an integral membrane protein. The amino-terminal end of the large α subunit is anchored by the β subunit. The bulk of the α protein protrudes into the cytosol. A large part of the sequence of the cytoplasmic region of the protein resembles a nucleic acid-binding protein, with many positive residues. This suggests the possibility that the SRP receptor recognizes the 7S RNA in the SRP.
There is a counterpart to SRP in bacteria, although it contains fewer components. E. coli contains a 4.5S RNA that associates with ribosomes and is homologous to the 7S RNA of the SRP. It associates with two proteins: Ffh is homologous to SRP54. FtsY is homologous to the α subunit of the SRP receptor. In fact, FtsY replaces the functions of both the α and β SRP subunits; its N-terminal domain substitutes for SRPβ in membrane targeting, and the C-terminal domain interacts with the target protein. The role of this complex is more limited than that of SRP-SRP receptor. It is probably required to keep some (but not all) secreted proteins in a conformation that enables them to interact with the secretory apparatus. This could be the original connection between protein synthesis and secretion; in eukaryotes the SRP has acquired the additional roles of causing translational arrest and targeting to the membrane.
Why should the SRP have an RNA component? The answer must lie in the evolution of the SRP: it must have originated very early in evolution, in an RNA-dominated world, presumably in conjunction with a ribosome whose functions were mostly carried out by RNA. The crystal structure of the complex between the protein-binding domain of 4.5S RNA and the RNA-binding domain of Ffh suggests that RNA continues to play a role in the function of SRP.
The 4.5S RNA has a region (domain IV) that is very similar to domain IV in 7S RNA (see Figure 8.24). Ffh consists of three domains (N, G, and M). The M domain (named for a high content of methionines) performs the key binding functions (Keenan et al., 1998). It has a hydrophobic pocket that binds the signal sequence of a target protein. The hydrophobic side chains of the methionine residues create the pocket by projecting into a cleft in the protein structure. Next to the pocket is a helix-turn-helix motif that is typical of DNA-binding proteins (see 12.12 Repressor uses a helix-turn-helix motif to bind DNA).
The crystal structure shows that the helix-loop-helix of the M domain binds to a duplex region of the 4.5S RNA in domain IV (Batey et al., 2000). The negatively charged backbone of the RNA is adjacent to the hydrophobic pocket. This raises the possibility that a signal sequence actually binds to both the protein and RNA components of the SRP. The positively charged sequences that start the signal sequence (see Figure 8.20) could interact with the RNA, while the hydrophobic region of the signal sequence could sit in the pocket.

GTP hydrolysis plays an important role in inserting the signal sequence into the membrane. Both the SRP and the SRP receptor have GTPase capability. The signal-binding subunit of the SRP, SRP54, is a GTPase. And both subunits of the SRP receptor are GTPases. All of the GTPase activities are necessary for a nascent protein to be transferred to the membrane. Figure 8.23 shows that the SRP starts out with GDP when it binds to the signal sequence. The ribosome then stimulates replacement of the GDP with GTP. The signal sequence inhibits hydrolysis of the GTP. This ensures that the complex has GTP bound when it encounters the SRP receptor.


For the nascent protein to be transferred from the SRP to the membrane, the SRP must be released from the SRP receptor. Figure 8.22 shows that this requires hydrolysis of the GTPs of both the SRP and the SRP receptor. The reaction has been characterized in the bacterial system, where it has the unusual feature that Ffh activates hydrolysis by FtsY, and FtsY reciprocally activates hydrolysis by Ffh (Powers and Walter, 1995).