Signal Sequences Of Secretory Proteins Research Articles

Silencing of Aberrant Secretory Protein Expression by Disease-Associated Mutations

Signal recognition particle (SRP) recognizes signal sequences of secretory proteins and targets them to the endoplasmic reticulum membrane for translocation. Many human diseases are connected with defects in signal sequences. The current dogma states that the molecular basis of the disease-associated mutations in the secretory proteins is connected with defects in their transport. Here, we demonstrate for several secretory proteins with disease-associated mutations that the molecular mechanism is different from the dogma. Positively charged or helix-breaking mutations in the signal sequence hydrophobic core prevent synthesis of the aberrant proteins and lead to degradation of their mRNAs. The degree of mRNA depletion depends on the location and severity of the mutation in the signal sequence and correlates with inhibition of SRP interaction. Thus, SRP protects secretory protein mRNAs from degradation. The data demonstrate that if disease-associated mutations obstruct SRP interaction, they lead to silencing of the mutated protein expression.

The Code for Directing Proteins for Translocation across ER Membrane: SRP Cotranslationally Recognizes Specific Features of a Signal Sequence

The signal recognition particle (SRP) cotranslationally recognizes signal sequences of secretory proteins and targets ribosome-nascent chain complexes to the SRP receptor in the endoplasmic reticulum membrane, initiating translocation of the nascent chain through the Sec61 translocon. Although signal sequences do not have homology, they have similar structural regions: a positively charged N-terminus, a hydrophobic core and a more polar C-terminal region that contains the cleavage site for the signal peptidase. Here, we have used site-specific photocrosslinking to study SRP–signal sequence interactions. A photoreactive probe was incorporated into the middle of wild-type or mutated signal sequences of the secretory protein preprolactin by in vitro translation of mRNAs containing an amber-stop codon in the signal peptide in the presence of the Nε-(5-azido-2 nitrobenzoyl)-Lys-tRNAamb amber suppressor. A homogeneous population of SRP–ribosome-nascent chain complexes was obtained by the use of truncated mRNAs in translations performed in the presence of purified canine SRP. Quantitative analysis of the photoadducts revealed that charged residues at the N-terminus of the signal sequence or in the early part of the mature protein have only a mild effect on the SRP–signal sequence association. However, deletions of amino acid residues in the hydrophobic portion of the signal sequence severely affect SRP binding. The photocrosslinking data correlate with targeting efficiency and translocation across the membrane. Thus, the hydrophobic core of the signal sequence is primarily responsible for its recognition and binding by SRP, while positive charges fine-tune the SRP–signal sequence affinity and targeting to the translocon.

Versatility of Trigger Factor Interactions with Ribosome-Nascent Chain Complexes

Trigger factor (TF) is the first molecular chaperone that interacts with nascent chains emerging from bacterial ribosomes. TF is a modular protein, consisting of an N-terminal ribosome binding domain, a PPIase domain, and a C-terminal domain, all of which participate in polypeptide binding. To directly monitor the interactions of TF with nascent polypeptide chains, TF variants were site-specifically labeled with an environmentally sensitive NBD fluorophore. We found a marked increase in TF-NBD fluorescence during translation of firefly luciferase (Luc) chains, which expose substantial regions of hydrophobicity, but not with nascent chains lacking extensive hydrophobic segments. TF remained associated with Luc nascent chains for 111 +/- 7 s, much longer than it remained bound to the ribosomes (t((1/2)) approximately 10-14 s). Thus, multiple TF molecules can bind per nascent chain during translation. The Escherichia coli cytosolic proteome was classified into predicted weak and strong interactors for TF, based on the occurrence of continuous hydrophobic segments in the primary sequence. The residence time of TF on the nascent chain generally correlated with the presence of hydrophobic regions and the capacity of nascent chains to bury hydrophobicity. Interestingly, TF bound the signal sequence of a secretory protein, pOmpA, but not the hydrophobic signal anchor sequence of the inner membrane protein FtsQ. On the other hand, proteins lacking linear hydrophobic segments also recruited TF, suggesting that TF can recognize hydrophobic surface features discontinuous in sequence. Moreover, TF retained significant affinity for the folded domain of the positively charged, ribosomal protein S7, indicative of an alternative mode of TF action. Thus, unlike other chaperones, TF appears to employ multiple mechanisms to interact with a wide range of substrate proteins.

Experimental confirmation of a key role for non-optimal codons in protein export

Non-optimal codons are defined by low usage and low abundance of corresponding tRNA, and have an established role in translational pausing to allow the correct folding of proteins. Our previous work reported a striking abundance of non-optimal codons in the signal sequences of secretory proteins exported via the sec-dependent pathway in Escherichia coli. In the current study the signal sequence of maltose-binding protein (MBP) was altered so that non-optimal codons were substituted with the most optimal codon from their synonymous codon family. The expression of MBP from the optimized allele ( malE-opt) was significantly less than wild-type malE. Expression of MBP from malE-opt was partially restored in a range of cytoplasmic and periplasmic protease deficient strains, confirming that reduced expression of MBP in malE-opt was due to its preferential degradation by cytoplasmic and periplasmic proteases. These data confirm a novel role for non-optimal codon usage in secretion by slowing the rate of translation across the N-terminal signal sequence to facilitate proper folding of the secreted protein.

The presence of a helix breaker in the hydrophobic core of signal sequences of secretory proteins prevents recognition by the signal-recognition particle in Escherichia coli.

Signal sequences often contain alpha-helix-destabilizing amino acids within the hydrophobic core. In the precursor of the Escherichia coli outer-membrane protein PhoE, the glycine residue at position -10 (Gly-10) is thought to be responsible for the break in the alpha-helix. Previously, we showed that substitution of Gly-10 by alpha-helix-promoting residues (Ala, Cys or Leu) reduced the proton-motive force dependency of the translocation of the precursor, but the actual role of the helix breaker remained obscure. Here, we considered the possibility that extension of the alpha-helical structure in the signal sequence resulting from the Gly-10 substitutions affects the targeting pathway of the precursor. Indeed, the mutations resulted in reduced dependency on SecB for targeting in vivo. In vitro cross-linking experiments revealed that the G-10L and G-10C mutant PhoE precursors had a dramatically increased affinity for P48, one of the constituents of the signal-recognition particle (SRP). Furthermore, in vitro cross-linking experiments revealed that the G-10L mutant protein is routed to the SecYEG translocon via the SRP pathway, the targeting pathway that is exploited by integral inner-membrane proteins. Together, these data indicate that the helix breaker in cleavable signal sequences prevents recognition by SRP and is thereby, together with the hydrophobicity of the signal sequence, a determinant of the targeting pathway.

SRP-dependent co-translational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein.

Recently it has been recognized that the signal recognition particle (SRP) of Escherichia coli represents a specific targeting device for hydrophobic inner membrane proteins. It has remained unclear, however, whether the bacterial SRP functions in concert with SecA, which is required for the translocation of secretory proteins across the inner membrane. Here, we have analyzed a hybrid protein constructed by fusing the signal anchor sequence of an SRP-dependent inner membrane protein (MtlA) to the mature part of an exclusively SecA-requiring secretory protein (OmpA). We show that the signal anchor sequence of MtlA confers the novel properties onto nascent chains of OmpA of being co-translationally recognized and targeted to SecY by SRP. Once targeted to SecY, ribosome-associated nascent chains of the hybrid protein, however, remain untranslocated unless SecA is present. These results indicate that SRP and SecA cooperate in a sequential, non-overlapping manner in the topogenesis of those membrane proteins which, in addition to a signal anchor sequence, harbor a substantial hydrophilic domain to be translocated into the periplasm.

Structure of a methionine-rich segment of Escherichia coli Ffh protein

The methionine-rich segments of the Ffh protein of Escherichia coli and its eukaryotic counterpart SRP54 are thought to bind signal sequences of secretory proteins. The structure of a chemically synthesized 25-residue-long peptide corresponding to one of the proposed methionine-rich amphiphilic helices of Ffh was determined in water and in aqueous trifluroethanol (TFE) solution using CD and NMR. An appreciable α-helix conformation exists even in water and this peptide assumes a stable α-helix along most of its length in aqueous TFE solution. It is clear that this segment of Ffh protein has a very strong propensity to form α-helical structure.

Transport of prepro-albumin into inverted vesicles prepared from the inner membrane of rat liver mitochondria

When inverted vesicles prepared from the inner membrane of rat liver mitochondria were incubated with prepro-rat serum albumin, considerable amounts of prepro-albumin and pro-albumin were recovered with the inverted vesicles re-isolated by centrifugation. Pro-albumin was resistant to trypsin, but prepro-albumin was completely digested by trypsin, indicating that prepro-albumin was transported into the vesicles and concomitantly converted to pro-albumin. This transport process required ATP, but not a membrane potential. These results suggest that some export machinery for a protein having an amino acid sequence in its N-terminal portion similar to the signal sequence of secretory protein exists in the inner mitochondrial membrane.

Role of the hydrophobic region of signal sequences in the targeting of proteins to membranes and translocation across the hydrophobic membrane barrier

Proteins destined for regions other than the cytoplasm in cells have to cross at least one membrane barrier before reaching their proper destination. Almost all such proteins are initially biosynthesized as precursors with signal sequences at the amino terminus. Signal sequences are essential and also sufficient for proteins to be targeted to membranes and also for translocation across membranes. One striking feature that is clearly evident amongst signal sequences of secretory proteins is a positively charged amino terminus followed by a region comprising 10–12 very hydrophobic amino acids. The structural and physico-chemical properties of signal sequences have been analysed. On the basis of the analyses it is proposed that the structural feature of a positively charged amino terminal region followed by a hydrophobic stretch of amino acids, rather than a conformational one, is recognised by components of the cells export machinery. It is also postulated that signal sequences insert in the lipid bilayer of the translocation competent membrane after targeting. The presence of the signal sequence results in the formation of local ‘defects’ in the bilayer which have a role in translocation of proteins across membranes.

Cell-free synthesis and segregation of beta 2-microglobulin.

beta2-Microglobulin has been synthesized in vitro by using a rabbit reticulocyte lysate system and mRNA from the mouse tumor cell line EL4. The molecule is synthesized as a precursor with an NH2-terminal extension of 19 amino acids: Ser-X-Ser-Val-X-Leu-Val-Phe-Leu-Val-Leu-Val-Ser-Leu-X-Gly-Leu-Tyr-X. The processing and segregation of this peripheral membrane protein are directly comparable to those of secretory proteins and integral membrane proteins: addition of dog pancreas microsomal membranes during translation caused conversion to the processed chain, but addition of membranes after synthesis did not; only the processed chain sedimented with the membrane vesicles and was protected from proteolysis by the vesicles; and processing of nascent beta 2-microglobulin was blocked by competitive inhibitors that prevent processing and segregation of secretory and integral membrane proteins. These results suggest that the signal sequences of secretory proteins, integral membrane proteins, and peripheral membrane proteins have a common function and a common receptor on the cytoplasmic face of dog pancreas microsomal membranes. This system also provides a means for studying in vitro the expression and function of the major histocompatibility antigens that are associated with beta 2-microglobulin on cell surfaces.