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The majority of outer membrane, periplasmic and some inner membrane proteins of Escherichia coli are synthesised with signal sequences which initiate the translocation process. It has been suggested that other polypeptide sequences within the mature protein carry additional information which determines the final localisation of the product. The aim of this project was to investigate the assembly into the outer membrane of the E. coli ferrichrome receptor protein, TonA. The tonA gene was subcloned onto pBR325 in order to maximise expression of this normally minor outer membrane protein. A study of the kinetics of assembly of TonA in a strain harbouring a multicopy plasmid carrying tonA revealed the occurrence of a processed assembly intermediate which separated with the soluble (cytoplasmic plus periplasmic) fraction of sonicated cells. The position and direction of transcription of tonA was deduced by Tn1000 mutagenesis followed by analysis of the resultant truncated TonA' polypeptides synthesised in vitro and in maxicells. All the TonA' polypeptides thus produced, even those with apparently small C-terminal deletions, fractionated with the sarkosyl soluble envelope material in maxicells (wild type TonA is sarkosyl insoluble), suggesting an important role for the C-terminus in assembly. A similar result was obtained when the tonA gene was truncated using an "oligo-stop translation" sequence. This eliminated the possibility that complete assembly of the TonA' polypeptides truncated by Tn1000 insertion was prevented by Tn1000 encoded sequences at their C-termini. Synthesis of the hybrid MalE-LacZ protein, 72-47, was demonstrated to inhibit the processing of TonA and several inner membrane proteins. Since this hybrid was already known to block the assembly and processing of periplasmic and outer membrane proteins, this result suggests that all three classes of exported protein share common steps in their assembly.

published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

Studies were conducted to determine the effects of cyclopropenoid fatty acids (CPFA) on the microsomal membrane of livers of rainbow trout (Salmo gairdneri). Slab and tube gel electrophoresis of microsomes from trout fed a CPFA diet (CPFA-microsomes) for varying time periods showed a decrease in the number of protein bands resolved in the high molecular weight region. This disappearance of high molecular weight proteins was not due to increased proteolysis in the CPFA-microsomes. Antibodies against whole microsomal protein from livers of trout fed 300 ppm CPFA were produced in rabbits. Microsomal proteins were first separated by polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose sheets (NC) and analyzed by the peroxidase-antiperoxidase (PAP) immunochemical staining procedure. Immunoabsorption of antisera directed against CPFA-microsomes by control-microsomes did not reveal any new proteins induced by the CPFA diets. However, the intensity of PAP staining was much greater in CPFA microsomes after immunoabsorption. Hydrolysis of phospholipids in the microsomal membrane by phospholipase A₂ failed to reveal any differences between control and CPFA fed trout. Proteolysis of microsomal membrane proteins had similar effects on NADPH cytochrome reductase and cytochrome P-450 activity on fish fed the different diets. PAGE analysis of these digests did show some differences in digested proteins between the control and CPFA group. These results may reflect a possible change in orientation of microsomal membrane proteins brought about by CPFA in the diet. Additional evidence for altered orientation of proteins was found with PAGE analysis of trypsin-digested microsomes. Moreover incubation of trypsin-digested microsomes with antisera and stained with PAP showed that proteolytic attack was different between control and CPFA microsomes. A final study with incubation of transferred proteins from control and CPFA-microsomes with antisera directed against purified cytochrome P-450 (P-450) and cytochrome P-448 (P-448) showed that CPFA had an effect on the concentration of P-448 but not P-450. / Graduation date: 1983

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