A substitution at His-120 in the LasA protease of Pseudomonas aeruginosa blocks enzymatic activity without affecting propeptide processing or extracellular secretion.

The LasA protease of Pseudomonas aeruginosa can degrade elastin and is an important contributor to the pathogenesis of this organism. LasA (20 kDa) is a member of the beta-lytic endopeptidase family of extracellular bacterial proteases, and it shows high-level staphylolytic activity. We sequenced the lasA gene from strain FRD1 and overexpressed it in Escherichia coli. The lasA gene encodes a precursor, known as pre-proLasA, of 45,582 Da. Amino-terminal sequence analysis allowed the identification of the signal peptidase cleavage site and revealed that the 31-amino-acid signal peptide was removed in E. coli. The remaining proLasA (42 kDa) did not undergo autoproteolytic processing and showed little staphylolytic activity. However, it was readily processed to a 20-kDa active staphylolytic protease by incubation with trypsin or with the culture filtrate of a P. aeruginosa lasAdelta mutant. Thus, removal of the propeptide (22 kDa) was required to convert proLasA into an active protease. Although LasA protease was critical for staphylolytic activity, other proteases like elastase were found to enhance staphylolysis. Under the control of an inducible trc promoter, lasA was overexpressed in P. aeruginosa and the processing intermediates were examined. Compared with wild-type cells, the overproducing cells accumulated more 42-kDa proLasA species, and the culture supernatants of the overproducing cells showed increased levels of active 20-kDa LasA protease. Small amounts of a 25-kDa extracellular LasA-related protein, which could represent a potential processing intermediate, were also observed. To better understand the structure-function relationships in LasA protease, we tested whether His-120-X-His-122 in the mature portion of LasA plays a role in activity. This motif and surrounding sequences are conserved in the related beta-lytic protease of Achromobacter lyticus. Oligonucleotide-directed mutagenesis was used to change His-120 to Ala-120, thus forming the lasA5 allele. The product of lasA5 expressed from the chromosome of P. aeruginosa was processed to a stable, secreted 20-kDa protein (designated LasA-H120A) which was devoid of staphylolytic activity. This suggests that His-120 is essential for LasA activity and favors the possibility that proLasA processing and secretion in P. aeruginosa can proceed via mechanisms which do not involve autoproteolysis.     

Role of Chitin-Binding Proteins in the Specific Attachment of the Marine Bacterium Vibrio harveyi to Chitin

We examined the mechanism of attachment of the marine bacterium Vibrio harveyi to chitin. Wheat germ agglutinin and chitinase bind to chitin and competitively inhibited the attachment of V. harveyi to chitin, but not to cellulose. Bovine serum albumin and cellulase do not bind to chitin and had no effect on bacterial attachment to chitin. These data suggest that this bacterium recognizes specific attachment sites on the chitin particle. The level of attachment of a chitinase-overproducing mutant of V. harveyi to chitin was about twice as much as that of the uninduced wild type. Detergent-extracted cell membranes inhibited attachment and contained a 53-kDa peptide that was overproduced by the chitinase-overproducing mutant. Three peptides (40, 53, and 150 kDa) were recovered from chitin which had been exposed to membrane extracts. Polyclonal antibodies raised against extracellular chitinase cross-reacted with the 53- and 150-kDa chitin-binding peptides and inhibited attachment, probably by sterically hindering interactions between the chitin-binding peptides and chitin. The 53- and 150-kDa chitin-binding peptides did not have chitinase activity. These results suggest that chitin-binding peptides, especially the 53-kDa chitin-binding peptide and chitinase and perhaps the 150-kDa peptide, mediate the specific attachment of V. harveyi to chitin.     

Solution structure, backbone dynamics and chitin binding of the anti-fungal protein from Streptomyces tendae TU901

AFP1 is a recently discovered anti-fungal, chitin-binding protein from Streptomyces tendae Tü901. Mature AFP1 comprises 86 residues and exhibits limited sequence similarity to the cellulose-binding domains of bacterial cellulases and xylanases. No similarity to the Cys and Gly-rich domains of plant chitin-binding proteins (e.g. agglutinins, lectins, hevein) is observed. AFP1 is the first chitin-binding protein from a bacterium for which anti-fungal activity was shown. Here, we report the three-dimensional solution structure of AFP1, determined by nuclear magnetic resonance spectroscopy. The protein contains two antiparallel beta-sheets (five and four beta-strands each), that pack against each other in a parallel beta-sandwich. This type of architecture is conserved in the functionally related family II of cellulose-binding domains, albeit with different connectivity. A similar fold is also observed in other unrelated proteins (spore coat protein from Myxococcus xanthus, beta-B2 and gamma-B crystallins from Bos taurus, canavalin from Jack bean). AFP1 is therefore classified as a new member of the betagamma-crystallin superfamily. The dynamics of the protein was characterized by NMR using amide 15N relaxation and solvent exchange data. We demonstrate that the protein exhibits an axially symmetric (oblate-like) rotational diffusion tensor whose principal axis coincides to within 15 degrees with that of the inertial tensor. After completion of the present structure of AFP1, an identical fold was reported for a Streptomyces killer toxin-like protein. Based on sequence comparisons and clustering of conserved residues on the protein surface for different cellulose and chitin-binding proteins, we postulate a putative sugar-binding site for AFP1. The inability of the protein to bind short chitin fragments suggests that certain particular architectural features of the solid chitin surface are crucial for the interaction.     

A conserved domain in arthropod cuticular proteins binds chitin

Many insect cuticular proteins include a 35-36 amino acid motif known as the R&R consensus. The extensive conservation of this region led to the suggestion that it functions to bind chitin. Provocatively, it has no sequence similarity to the well-known cysteine-containing chitin-binding domain found in chitinases and some peritrophic membrane proteins. Using fusion proteins expressed in E. coli, we show that an extended form of the R&R consensus from proteins of hard cuticles is necessary and sufficient for chitin binding. Recombinant AGCP2b, a putative cuticular protein from the mosquito Anopheles gambiae, was expressed in E. coli and the purified protein shown to bind to chitin beads. A stretch of 65 amino acids from AGCP2b, including the R&R consensus, conferred chitin binding to glutathione-S-transferase (GST). Directed mutagenesis of some conserved amino acids within this extended R&R consensus from hard cuticle eliminated chitin binding. Thus arthropods have two distinct classes of chitin binding proteins, those with the chitin-binding domain found in lectins, chitinases and peritrophic membranes (cysCBD) and those with the cuticular protein chitin-binding domain (non-cysCBD).     

Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity.

A 22-kilodalton protein purified from the culture supernatant fraction of Pseudomonas aeruginosa (strains PA220 and PAO1) was found to enhance the elastolytic activity of purified P. aeruginosa elastase. N-terminal sequence analysis identified the protein as a fragment of the lasA gene product (P.A. Schad and B.H. Iglewski, J. Bacteriol. 170:2784-2789, 1988). However, comparative analysis with the reported LasA sequence indicated that the purified LasA fragment is longer than the deduced sequence reported. The purified LasA fragment had minimal elastolytic and proteolytic activity and did not enhance the proteolytic activity of purified elastase, yet enhanced the elastolytic activity more than 25-fold. The LasA fragment was found to also enhance the elastolytic activities of thermolysin, human neutrophil elastase, and proteinase K. The results presented here suggest that the LasA protein interacts with the elastin substrate rather than modifying elastase.     

Cloning and characterization of elastase structural gene from Pseudomonas aeruginosa IFO 3455

An 8.3 Kb DNA fragment was cloned from Pseudomonas aeruginosa IFO 3455. This fragment-containing Escherichia clone, pEL2, produced a high level of elastase activity. A smaller EcoRI-KpnI fragment was subcloned into pUC118 and E. coli HB101 was transformed with the plasmid. A deletion mutant clone was also constructed in the same bacteria. These deletion mutants were tested for elastase activity and it became clear that the full length of the elastase gene was 1.0-1.3 Kb. DNA sequencing analysis revealed that this DNA fragment contains the DNA sequence coding N-terminal amino acid sequence of the elastase protein.     

Characterization of Pseudomonas aeruginosa chitinase, a gradually secreted protein.

The gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene, chiC, coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of Serratia marcescens, ChiA of Vibrio harveyi, and ChiD of Bacillus circulans and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, but not on p-nitrophenyl-beta-D-N-acetylglucosamine, indicating that it is an endochitinase. Expression of the chiC gene appears to be regulated by the quorum-sensing system of P. aeruginosa, since this gene was not expressed in a lasIR vsmI mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.     

A structural and functional analysis of the docking protein. Characterization of active domains by proteolysis and specific antibodies

Docking protein is a 73-kDa integral membrane protein of the rough endoplasmic reticulum. It is essential for translocation of nascent secretory proteins into the lumen of the endoplasmic reticulum. Monoclonal and polyclonal antibodies have been generated which, in conjunction with limited proteolysis, have been used to characterize several subspecies of docking protein. These proteolytic fragments have been analyzed with respect to the various functions ascribed to docking protein which can be assayed in vitro. Proteolytic digestion of membrane-associated or of affinity-purified intact docking protein showed that: elastase cleavage generates a 59-kDa soluble fragment and one of 14 kDa which contains the membrane anchoring domain; trypsin as well as endogenous proteolysis generates a 46-kDa fragment, leaving a 27-kDa domain containing the membrane anchor. This 27-kDa fragment can be reduced to a 13- and a 14-kDa piece by elastase digestion. The characteristics of these various subspecies were examined. The 59-kDa soluble fragment, which can reconstitute full translocation activity to docking protein-depleted microsomes (Meyer, D. I., and Dobberstein, B. (1980) J. Cell Biol. 87, 503-508) was capable of releasing a signal recognition particle-mediated translation arrest. The 46-kDa fragment was neither able to reassociate with nor to reconstitute the activity of docking protein-depleted microsomes. Moreover this fragment was unable to release a signal recognition particle-mediated arrest. This suggests that the 13-kDa fragment (the difference between 46 and 59 kDa) is both essential for association with the membrane, and for the release of translation arrests.     

Cloning and Analysis of the Capsid Morphogenesis Genes of Pseudomonas aeruginosa Bacteriophage D3: Another Example of Protein Chain Mail?

The terminal DNA restriction fragments (PstI-D and -B) of Pseudomonas aeruginosa bacteriophage D3 were ligated, cloned, and sequenced. Of the nine open reading frames in this 8.3-kb fragment, four were identified as encoding large-subunit terminase, portal, ClpP protease, and major head proteins. The portal and capsid proteins showed significant homology with proteins of the lambdoid coliphage HK97. Phage D3 was purified by CsCl equilibrium gradient centrifugation (rho = 1.533 g/ml), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed six proteins with molecular masses of 186, 91, 79, 70, 45, and 32 kDa. The pattern was unusual, since a major band corresponding to the expected head protein (43 kDa) was missing and a significant amount of the protein was retained in the stacking gel. The amino terminus of the 186-kDa protein was sequenced, revealing that the D3 head is composed of cross-linked 31-kDa protein subunits, resulting from the proteolysis of the 43-kDa precursor. This is identical to the situation observed with coliphage HK97.     

The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation.

The mature form of chitinase A1 from Bacillus circulans WL-12 comprises a C-terminal domain, two type III modules (domains), and a large N-terminal domain which contains the catalytic site of the enzyme. In order to better define the roles of these chitinase domains in chitin degradation, modified chiA genes encoding various deletions of chitinase A1 were constructed. The modified chiA genes were expressed in Escherichia coli, and the gene products were analyzed after purification by high-performance liquid chromatography. Intact chitinase A1 specifically bound to chitin, while it did not show significant binding activity towards partially acetylated chitosan and other insoluble polysaccharides. Chitinases lacking the C-terminal domain lost much of this binding activity to chitin as well as colloidal chitin-hydrolyzing activity. Deletion of the type III domains, on the other hand, did not affect chitin-binding activity but did result in significantly decreased colloidal chitin-hydrolyzing activity. Hydrolysis of low-molecular-weight substrates, soluble high-molecular-weight substrates, and insoluble high-molecular-weight substrates to which chitinase A1 does not bind were not significantly affected by these deletions. Thus, it was concluded that the C-terminal domain is a chitin-binding domain required for the specific binding to chitin and that this chitin-binding activity is important for efficient hydrolysis of the sufficiently acetylated chitin. Type III modules are not directly involved in the chitin binding but play an important functional role in the hydrolysis of chitin by the enzyme bound to chitin.     

Purification and characterization of LasD: a second staphylolytic proteinase produced by Pseudomonas aeruginosa

We have previously described studies of a 22 kDa active fragment of the LasA proteinase. In follow-up studies of LasA, we have discovered the separate existence of a 23 kDa proteinase which shares many of the enzymatic properties of LasA, including the ability to lyse heat-killed staphylococoi. However, this apparent serine proteinase, which we designate LasD, is distinct from the 22 kDa active LasA protein for the following reasons: (i) the N-terminal sequence of LasD shares no homology with LasA or the LasA precursor sequence; (ii) Pseudomonas aeruginosa LasA mutant strains AD1825 and FRD2128 do not produce LasA yet produce LasD; and (iii) specific antibodies to each proteinase do not show any cross-reactivity. LasD appears to be produced as a 30 kDa protein, which is possibly cleaved to produce a 23 kDa active fragment. The purified LasD fragment (23 kDa) shows strong staphylolytic activity only at higher pH conditions, while LasA exhibits staphylolytic activity over a broad pH range, in addition to their ability to cleave at internal diglycine sites, both the LasD and LasA endoproteinases efficiently cleave β-casein.     

A class I chitinase from soybean seed coat.

Protein extracts from soybean (Glycine max [L.] Merr) seed hulls were fractionated by isoelectric focusing and SDS-PAGE analysis and components identified by peptide microsequencing. An abundant 32 kDa protein possessed an N-terminal cysteine-rich hevein domain present in class I chitinases and in other chitin-binding proteins. The protein could be purified from seed coats by single step binding to a chitin bead matrix and displayed chitinase activity by an electrophoretic zymogram assay. The corresponding cDNA and genomic clones for the chitinase protein were isolated and characterized, and the expression pattern determined by RNA blot analysis. The deduced peptide sequence of 320 amino acids included an N-terminal signal peptide and conserved chitin-binding and catalytic domains interspaced by a proline hinge. An 11.3 kb EcoRI genomic fragment bearing the 2.4 kb chitinase gene was fully sequenced. The gene contained two introns and was flanked by A+T-rich tracts. Analysis by DNA blot hybridization showed that this is a single or low copy gene in the soybean genome. The chitinase is expressed late in seed development, with particularly high expression in the seed coat. Expression was also evident in the late stages of development of the pod, root, leaf, and embryo, and in tissues responding to pathogen infection. This study further illustrates the differences in protein composition of the various seed tissues and demonstrates that defence-related proteins are prevalent in the seed coat.     

Quality control of Photosystem II: cleavage and aggregation of heat-damaged D1 protein in spinach thylakoids

Moderate heat stress (40 degrees C, 30 min) on spinach thylakoids induced cleavage of the D1 protein, producing an N-terminal 23-kDa fragment, a C-terminal 9-kDa fragment, and aggregation of the D1 protein. A homologue of Arabidopsis FtsH2 protease, which is responsible for degradation of the damaged D1 protein, was abundant in the stroma thylakoids. Two processes occurred in the thylakoids in response to heat stress: dephosphorylation of the D1 protein in the stroma thylakoids, and aggregation of the phosphorylated D1 protein in the grana. Heat stress also induced the release of the extrinsic PsbO, P and Q proteins from Photosystem II, which affected D1 degradation and aggregation significantly. The cleavage and aggregation of the D1 protein appear to be two alternative processes influenced by protein phosphorylation/dephosphorylation, distribution of FtsH, and intactness of the thylakoids.     

Antifungal thiopeptide cyclothiazomycin B1 exhibits growth inhibition accompanying morphological changes via binding to fungal cell wall chitin

Cyclothiazomycin B1 (CTB1) is an antifungal cyclic thiopeptide isolated from the culture broth of Streptomyces sp. HA 125-40. CTB1 inhibited the growth of several filamentous fungi including plant pathogens along with swelling of hyphae and spores. The antifungal activity of CTB1 was weakened by hyperosmotic conditions, and hyphae treated with CTB1 burst under hypoosmotic conditions, indicating increased cell wall fragility. CTB1-sensitive fungal species contain high levels of cell wall chitin and/or chitosan. Unlike nikkomycin Z, a competitive inhibitor of chitin synthase (CHS), CTB1 did not inhibit CHS activity. Although CTB1 inhibited CHS biosynthesis, the same result was also obtained with a non-specific proteins inhibitor, cycloheximide, which did not reduce cell wall rigidity. These results indicate that the primary target of CTB1 is not CHS, and we concluded that CTB1 antifungal activity was independent of this sole inhibition. We found that CTB1 bound to chitin but did not bind to β-glucan and chitosan. The results of the present study suggest that CTB1 induces cell wall fragility by binding to chitin, which forms the fungal cell wall. The antifungal activity of CTB1 could be explained by this chitin-binding ability.     

Production and processing of a 59-kilodalton exochitinase during growth of Streptomyces lividans carrying pCHIO12 in soil microcosms amended with crab or fungal chitin.

Streptomyces lividans (pCHIO12), which carries the previously cloned Streptomyces olivaceoviridis exo-chiO1 gene on a multicopy vector, secretes a 59-kDa exochitinase, consisting of a catalytic domain (40 kDa), a central fibronectin type III-like module, and a chitin-binding domain (12 kDa). The propagation rate of S. lividans (pCHIO12) was higher in soil microcosms amended with fungal mycelia than in those containing crab chitin. Comparative biochemical and immunological studies allowed the following conclusions to be drawn. Within soil microcosm systems amended with crab shell chitin or chitin-containing Aspergillus proliferans mycelia, the strain expressed the clones exo-chiO1 gene and produced high quantities of a 59-kDa exochitinase. The enzyme was preferentially attached via its binding domain to the pellet from soil or liquid cultures. In contrast, truncated forms of 47, 40, and 25 kDa could be easily extracted from soil. The relative proportions of the 59-kDa enzyme and its truncated forms varied depending on the source of chitin and differed in soil and in liquid cultures.     

Induction of Chitin-Binding Proteins during the Specific Attachment of the Marine Bacterium Vibrio harveyi to Chitin

Previous work has shown that attachment of Vibrio harveyi to chitin is specific and involves at least two chitin-binding peptides. However, the roles and regulation of these chitin-binding peptides in attachment are still unclear. Here we show that preincubation with the oligomeric sugars composing chitin stimulated chitinase activity, cellular attachment to chitin, and production of chitin-binding peptides. One of these peptides, a 53-kDa peptide, is produced constitutively and appears to mediate initial attachment to chitin. Synthesis of another peptide, a 150-kDa chitin-binding peptide, is induced by chitin and thus may be involved in time-dependent attachment. Coordinated regulation of attachment and degradation of chitin may give bacteria like V. harveyi a selective advantage over other bacteria in nutrient-poor aquatic environments.     

Quality control of Photosystem II: Cleavage and aggregation of heat-damaged D1 protein in spinach thylakoids

Moderate heat stress (40 °C, 30 min) on spinach thylakoids induced cleavage of the D1 protein, producing an N-terminal 23-kDa fragment, a C-terminal 9-kDa fragment, and aggregation of the D1 protein. A homologue of Arabidopsis FtsH2 protease, which is responsible for degradation of the damaged D1 protein, was abundant in the stroma thylakoids. Two processes occurred in the thylakoids in response to heat stress: dephosphorylation of the D1 protein in the stroma thylakoids, and aggregation of the phosphorylated D1 protein in the grana. Heat stress also induced the release of the extrinsic PsbO, P and Q proteins from Photosystem II, which affected D1 degradation and aggregation significantly. The cleavage and aggregation of the D1 protein appear to be two alternative processes influenced by protein phosphorylation/dephosphorylation, distribution of FtsH, and intactness of the thylakoids.     

Expression inEscherichia coliof Y5-Mutant and N-terminal Domain-Deleted DNA Gyrase B Proteins Affects Strongly Plasmid Maintenance

Escherichia coliDNA gyrase B subunit (GyrB) is composed of a 43-kDa N-terminal domain containing an ATP-binding site and a 47-kDa C-terminal domain involved in the interaction with the gyrase A subunit (GyrA). Site-directed mutagenesis was used to substitute, in both the entire GyrB subunit and its 43-kDa N-terminal fragment, the amino acid Y5 by either a serine (Y5S) or a phenylalanine residue (Y5F). Under standard conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transformed with the wild-type plasmid. This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-terminal 43-kDa mutant plasmids were used. Examination of the plasmid content of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number. By repressing tightly the promoter activity encoded by these expression vectors during cell growth, it was possible to restore the normal level of the mutant GyrB encoding plasmids in the transformed bacteria. Treatment with chloramphenicol before protein induction enabled large overexpression of the GyrB mutant Y5F and Y5S proteins. In addition, the decrease in plasmid copy number was also observed when the 47-kDa C-terminal fragment of the GyrB subunit was expressed in bacteria grown under standard culture conditions. Analysis of DNA supercoiling and relaxation activities in the presence of GyrA demonstrated that purified Y5-mutant GyrB proteins were deficient for ATP-dependent gyrase activities. Taken together, these results show that Y5F and Y5S mutant GyrB proteins, but not the corresponding 43-kDa N-terminal fragments, competein vivowith the bacterial endogenous GyrB subunit of DNA gyrase, thereby reducing the plasmid copy number in the transformed bacteria by probably acting on the level of negative DNA supercoilingin vivo.This competition could be mediated by the presence of the intact 47-kDa C-terminal domain in the Y5F and Y5S mutant GyrB subunits. This study demonstrates also that the amino acid Y5 is a crucial residue for the expression of the gyrase B activityin vivo.Thus, ourin vivoapproach may also be useful for detecting other important amino acids for DNA gyrase activity, as mutations affecting the ATPase activity or the GyrB/GyrB or GyrB/GyrA protein interactions.     

Disassembly and domain structure of the proteins in the signal-recognition particle

The signal-recognition particle (SRP) is a ribonucleoprotein (RNP) complex consisting of six different polypeptide chains and a 7SL RNA. It participates in initiating the translocation of proteins across the membrane of the endoplasmic reticulum. SRP was disassembled in 2 M KCl into three components, one RNP composed of 7SL RNA and the 54-kDa and 19-kDa proteins, and two heterodimers consisting of the 72/68-kDa and the 14/9-kDa proteins respectively. The 54-kDa protein could be released from the RNP subparticle by chromatography on DEAE-Sepharose in Mg2+-depleted buffer, while the 19-kDa protein remained bound to the 7SL RNA. The domain structure of SRP proteins was probed by using mild elastase treatment and protein-specific antibodies. It was found that the 72, 68, 54 and 19-kDa SRP proteins were proteolytically processed in distinct steps. Most remarkably a protein fragment of 55-kDa, generated from the 72-kDa SRP protein, and a 35-kDa fragment from the 54-kDa SRP protein were both released from the RNP particle. Fragments generated from the 68-kDa protein and detectable with the anti-(68-kDa protein) antibody remained associated with the RNP particle. Cleavage of the SRP proteins by elastase at 2.5 micrograms/ml resulted in partial loss of activity, while 10 micrograms/ml caused complete inactivation of the particle. Neither the elongation arrest of IgG light chain nor its translocation across SRP-depleted microsomal membranes was promoted. The implications of these results on the possible interaction between the SRP subunits are discussed.