The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coli which, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4.5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and ⌬H ϩ . In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.

In eukaryotes, the cotranslational targeting of proteins to the endoplasmic reticular membrane is initially mediated by the signal-recognition particle (SRP), a ribonucleoprotein complex consisting of the 7SL RNA and six protein subunits. Since the discovery of sequence homology between (a) the Escherichiu coli 4.5s RNA (Ffs) and 7SL RNA, and (b) the E. coli P48 (Ffh) and SRP 54-kDa subunit, more evidence has been obtained that E. coli also possesses an SRP-type pathway that acts in the translocation of secreted proteins. Such a pathway could possibly be involved in the cotranslational integration of hydrophobic membrane proteins that cannot be effectively targeted post-translationally due to folding and aggregation. In this study, we report that disruption of the E. coli SRP complex with a dominant lethal 4.5s RNA mutant iiz vivo prevents functional membrane integration of the E. coli lactose permease (Lacy). Likewise, depletion of the P48 (Ffh) protein also results in a decrease in the amount of functional Lacy inserted into the E. coli plasma membrane. In direct contrast, inhibition of SecA function does not affect L a c y integration. These results suggest a major function of the bacterial SRP in the targeting and subsequent integration of hydrophobic membrane proteins as opposed to SecA mediating the posttranslational targeting of secretory proteins.Keywords: lactose permease; protein translocation ; Escherichia coli; membrane integration.Most of the components involved in the translocation of proteins into and across the Esclzerichia coli cytoplasmic membrane have been identified by the isolation of conditional lethal mutations showing generalised secretion defects (SecA, B, D-G, Y) and their role in the translocation of exported proteins has been analysed by the use of in vitro systems reconstituted from purified Sec proteins 11 -71. Until recently, this was thought to be the only pathway of protein export in E. coli. However, discovery that the E. coli genes for the 4.5s RNA Ws), P48 m h ) , and FtsY are the bacterial homologues to the eukaryotic 7SL RNA, 54-kDa signal-recognition particle (SRP) subunit, and SRP receptor, respectively [8-lo], and subsequent demonstratiori of their possible role in protein translocation [ll -171 have led to suggestions that another evolutionary-conserved pathway exists.In contrast to exported proteins, very little is known regarding the targeting and imertion of integral membrane proteins in E. coli. These proteins are inserted without the cleavage of a signal sequence and whether or not the authentic membrane protein requires the components of the Sec machinery s e e m to depend on the protein under study [18][19][20]. In contrast, most eukaryotic membrane proteins have been shown to strictly depend on the SRP for membrane integration [2].The lactose permease (Lacy) of E. coli is a widely studied polytopic transmembrane protein, but contradictory results have been obtained concerning its Sec dependence. h z vivo pulse labeling of overproduced L a c y and demonstration ...

A Leishmania donovani promastigote cDNA library was screened with serum obtained from a patient infected with visceral leishmaniasis. Sequence analysis of a clone obtained from this library revealed that the 600-bp insert corresponded to the carboxy-terminal region of an antigen related to the 70-kDa heat-shock protein family. The full-length sequence of the corresponding gene (1 959 nucleotides) was determined after isolation of genomic clones. Genes encoding the antigen are present on a single chromosome as a series of approximately twelve 3.7-kb direct tandem repeats. The antigen can be identified as a 70-kDa heat-shock cognate protein by virtue of its molecular mass, sequence and constitutive expression during heat shock. It is expressed at all stages of the parasite life-cycle. Antibodies against the Lgtl 1 fusion protein were detected in more than 50% of serum samples obtained from patients with visceral leishmaniasis, but were not detected in sera from patients with cutaneous leishmaniasis or Chagas' disease.

Using purified bacterially expressed herpes simplex virus type 1 ribonucleotide reductase large subunit (R 1) and the proteolytic enzymes chymotrypsin and trypsin, we have generated stable N-terminal truncations. Chymotrypsin removes 246 amino acids from the amino terminus to produce a fragment (dN246R1) which retains full enzymic activity and affinity for the small subunit (R2). Treatment of R1 with trypsin produces a 120K protein and a cleavage at amino acid residue 305 to produce a fragment (dN305R1) which remains associated with a 33K N-terminal polypeptide. Although this 33K-dN305R1 complex retains full binding affinity for R2 its reductase activity is reduced by approximately 50 %. Increasing the concentration of trypsin removes the 33K N-terminal polypeptide resulting in dN305R1 which, when bound to R2, has full ribonucleotide reductase activity. Like R1, dN246R1 and dN305R1 each exist as dimers showing that the first 305 amino acids of R1 are not necessary for dimer formation. These results indicate that, in structural studies of subunit interaction, dN246R1 or dN305R1 can be considered as suitable replacements for intact R1.

We have shown that a member of the 70-kDa heat shock protein (Hsp7O) family is a major target of the humoral immune response during Leishmania donovani infection. A recombinant fusion protein was recognized by sera from 92% (35 of 38) of patients with visceral leishmaniasis, including representatives from each of the major regions where it is endemic. Serological analysis of recombinant Hsp7O, expressed by a series of deletion constructs, identified the carboxy-terminal region as the immunodominant site. This region, which is the most evolutionarily divergent part of the molecule, was recognized by all sera from patients with visceral leishmaniasis which exhibited an anti-Hsp7O response. Purified recombinant L. donovani Hsp7O was not recognized by sera from patients with cutaneous leishmaniasis, Chagas' disease, leprosy, malaria, or schistosomiasis. To determine the regions involved in antibody recognition, a series of overlapping peptides were synthesized on polyethylene pins by the Pepscan method, and a hexamer, EADDRA, was identified by the visceral leishmaniasis serum samples as an immunodominant B-cell epitope.