Precursors to two periplasmic proteins and one outer membrane protein were synthesized in a membrane-free extract from Escherichia coli programmed with plasmid DNA. In the presence of inverted plasma membrane vesicles from E. coli up to 25% of the precursor molecules were converted into their mature forms. Using externally added proteinase K as a probe, we found the processed proteins segregated within the membrane vesicles. By the same criteria, a small amount of each precursor also proved to be translocated, indicating that translocation and signal sequence cleavage are not necessarily coupled processes. Furthermore, we present conclusive evidence that the translocation step can occur post-translationally even as late as 60 min after the beginning of translation.

https://www.pnas.org/content/pnas/81/23/7421.full.pdf

In vitro translocation of bacterial proteins across the plasma membrane of Escherichia coli

Importance of the propeptide sequence of human preproparathyroid hormone for signal sequence function

Publisher Summary Secretory proteins are synthesized on ribosomes located in the cytoplasm and are released from the cell as a fairly homogeneous, highly concentrated population of molecules. Secretory proteins can be localized in the lumen of the endoplasmic reticulum (ER) very early in their existence. Translocation of secretory proteins from the cytoplasmic to the luminal side of the rough ER membrane is an essential feature of the transport of these molecules. The information for the association of the mRNAs encoding secretory proteins to the membrane is contained within secretory protein mRNAs itself. mRNA encoding a secretory protein contains the signal for its ultimate association with the membrane. All secretory protein mRNAs encode a transient N-terminal peptide. This short amino acid sequence provides a signal, which results in the formation of membrane-bound polysomes and, ultimately, the translocation across the membrane. This chapter presents the results of studies, which deal with various individual aspects of protein translocation. It also describes signal sequences, signal recognition particle, docking protein, ribosome binding, signal peptidase, and the cotranslational covalent modifications, which are carried out in the rough ER.

Transfer of secretory proteins through the membrane of the endoplasmic reticulum.

Many proteins that are synthesized in the cytoplasm of cells are ultimately found in non-cytoplasmic locations. The correct targeting and transport of proteins must occur across bacterial cell membranes, the endoplasmic reticulum membrane, and those of mitochondria and chloroplasts. One unifying feature among transported proteins in these systems is the requirement for an amino-terminal targeting signal. Although the primary sequence of targeting signals varies substantially, many patterns involving overall properties are shared. A recent surge in the identification of components of the transport apparatus from many different systems has revealed that these are also closely related. In this review we describe some of the key components of different transport systems and highlight these common features.

Protein transport via amino-terminal targeting sequences: common themes in diverse systems.

Publisher Summary This chapter discusses the structure and expression of the human parathyroid hormone gene. Parathyroid hormone (PTH) stimulates the release of calcium from bone, the reabsorption of calcium from the kidney's glomerular filtrate, and through the 1-hydroxylation of 25-hydroxyvitamin D, the absorption of calcium across the intestinal mucosa. However, a negative feedback loop between calcium and PTH tends to stabilize the blood calcium within a narrow range. The pathway leading to PTH synthesis, like that leading to synthesis of other secreted peptides, involves a series of proteolytic cleavages of a large precursor. The gene encoding PTH like most other genes is flanked by DNA sequences controlling the gene's expression. The transcribed region contains two types of sequences—exons and introns, or intervening sequences. The isolation of the PTH gene and determination of its detailed structure required the screening of a gene library using recombinant DNA techniques. By comparing the DNA sequences of cyclic DNA with the sequences of genomic DNA, it was possible to deduce which genomic sequences are found in the mature mRNA (exons) and which sequences are removed (introns). More recent studies using radioactive PTH genes to bind to chromosomal squashes (in situ hybridization) have confirmed the gene's assignment to the short arm of chromosome 11 and further suggest that the gene is located near the end of the chromosome in 11p15.

Structure and expression of the human parathyroid hormone gene.

Eucaryotic endoplasmic reticulum and bacterial inner cell membranes use strikingly similar mechanisms to transport proteins from one cellular compartment to another. Using a mammalian linked transcription-translation system, we show here that canine pancreatic microsomes accurately cleave off the signal sequence of β-lactamase, a secreted bacterial protein. We suggest that the apparent differences between bacterial and eukaryotic protein transport may involve only minor modulations of a profoundly conserved mechanism.

/pdf/transport-and-cleavage-of-bacterial-pre-b-lactamase-by-4jutnr75wk.pdf

BJ Fennick

Papers

Transport and cleavage of bacterial pre-β-lactamase by mammalian microsomes