Target-directed proteolysis at the ribosome

Abstract

Target directed proteolysis allows specific processing of proteins in vivo. This method uses tobacco etch virus (TEV) NIa protease that recognizes a seven-residue consensus sequence. Because of its specificity, proteins engineered to contain a cleavage site are proteolysed, whereas other proteins remain unaffected. Therefore, this approach can be used to study the structure and function of target proteins in their natural environment within living cells. One application is the conditional inactivation of essential proteins, which is based on the concept that a target containing a recognition site can be inactivated by coexpressed TEV protease. We have previously identified one site in the secretion factor SecA that tolerated a TEV protease site insert. Coexpression of TEV protease in the cytoplasm led to incomplete cleavage and a mild secretion defect. To improve the efficiency of proteolysis, TEV protease was attached to the ribosome. We show here that cleaving SecA under these conditions is one way of increasing the efficiency of target directed proteolysis. The implications of recruiting novel biological activities to ribosomes are discussed. membrane protein insertion ͉ ribosome attachment ͉ transport P rotein translocation across the cytoplasmic membrane is a highly conserved process. Common features include the recognition of targeting signals of the secretory protein by dedicated cellular factors on the cis side of a membrane, a heterooligomeric channel that gates across and into the membrane, a peripherally associated component providing energy and a folding system on the trans side of the membrane (for review, see ref. 1). In bacteria, classical secretory proteins are synthesized with a N-terminal signal peptide. These preproteins are normally transferred across the cytoplasmic membrane in an extended conformation. The translocation channel or Sec machinery is a well studied heterooligomeric complex consisting of at least three integral membrane proteins, SecY, SecE, and SecG, and the peripherally bound cytoplasmic ATPase SecA (2, 3).The posttranslational targeting pathway of soluble proteins is well understood. In the cytoplasm, cleavable signal sequences are recognized by the chaperone SecB and the essential SecA protein. SecA is a multifunctional component that acts as a targeting factor, provides energy for translocation through its ATPase activity, and acts as a chaperone excluding nonsecretory proteins from translocation (4-6). Targeting of cytoplasmic membrane proteins depends on transmembrane segments that function as internal and noncleavable signal and stop-transfer sequences. Therefore, transmembrane segments do not only function in targeting but are also involved in determining the transmembrane topology (7-10). Recent evidence suggests that targeting of integral membrane proteins is cotranslational, involving the components of the signal recognition particle (SRP) system. This targeting includes the SRP itself, composed of the Ffh protein and the 4.5S RNA, and the SRP receptor FtsY (reviewe...

Depletion of the Signal Recognition Particle Receptor Inactivates Ribosomes in Escherichia coli

“…The level of depletion, however, probably directly influences the subsequent physiological responses and, thus, could in principle explain the difference between the in vivo and the in vitro observations. That the level of FtsY depletion might influence the subsequent protein synthesis defect is also in line with the observation that a specific inhibition of membrane proteins has not been observed in all in vivo studies using FtsY-depleted cells (20,25,45). Still, we currently cannot exclude the possibility that the strong effects we observe in our study conceal a more subtle response of E. coli that impairs the synthesis of membrane proteins more severely than the synthesis of soluble proteins.…”

“…A similar downregulation of ribosomal proteins and upregulation of chaperones was observed in S. mutans (23). Depletion of the SRP components in E. coli also leads to an induction of the 32 -regulated stress response, e.g., the induction of chaperones like DnaK and GroEL and proteases like Lon and ClpQ (9,25), but the effect on protein synthesis is largely unknown.…”

Discovery and Validation of New Regulatory RNA Elements in Chlamydia trachomatis

“…Total digestion of the fusion protein was evident for clones having tig144TEVsh (lane 3, 5). This result is in complete agreement with Henrichs et al, 2005.…”

“…The tethered TEV protease gained access to the nascent polypeptide as it exited the ribosome before folding occurs. The technique was successful at getting complete cleavage of the target protein (Henrichs et al, 2005). The tethering motive was cloned from an E. coli trigger factor (tig).…”

“…The domain that retained the ribosomal tethering activity was the N terminal 144 residues. Henrichs et al, 2005 fused this domain to the N terminus of TEV protease and the result was complete cleavage of SecA195. We adopted the same strategy and fused tig144 to the TEVsh protease.…”