No description available.

No description available.

No description available.

There are six known secretion systems in Gram negative bacteria, referred to as Type 1 to Type 6 respectively, which are dedicated to moving substrate across the outer membrane. Secretion systems are broadly separated into those that move their substrate across the cell envelope in a single translocation event (one-step systems), and those that are dependent on the Sec or Tat machineries for export to the periplasm (two-step systems). Serratia marcescens is an important opportunistic human pathogen and has gathered a lot of interest due to its repertoire of secreted proteins. These include the haem-scavenging protein HasA, which is secreted by a Type 1 secretion system, and the cytotoxic haemolysin ShlA, which is secreted as part of a two-partner Type 5 secretion system. Serratia marcescens also encodes a Type 6 secretion system, which is known to translocate at least six effector molecules directly into other bacterial target cells. Serratia marcescens is a model organism in terms of its ability to degrade the quite intractable polymer chitin, for which it produces three chitinase enzymes ChiA, ChiB, ChiC and a chitin-binding protein Cbp21, which hydrolyse the ß-1,4 link in the chitin chain and promote binding of chitinase to the chitin substrate respectively. These chitinolytic enzymes are utilised by S. marcescens for both basic physiology and also in pathogenesis. In this work, genetic, biochemical and proteomic approaches identified, for the first time, genes that are essential for the secretion of all three chitinases as well as Cbp21. A genetic screen identified genes encoding a holin-like membrane protein (ChiW) and a putative L-alanyl-D-glutamate endopeptidase (ChiX). Subsequent quantitative proteomics experiments and biochemical analyses established that ChiW and ChiX were required for secretion of the entire chitinolytic machinery. Chitinase secretion was observed to be blocked at a late stage in the mutant strains as normally secreted enzymes were found to accumulate in the periplasm, thus implicating ChiW and ChiX in a novel outer membrane protein translocation process. It is proposed that the bacterial genome-encoded holin-like protein and endopeptidase identified represent a putative secretion system utilised by Gram-negative bacteria. In addition to this, genes encoding the chitinolytic machinery and the putative secretion apparatus were shown to be bimodally regulated and co-ordinately expressed.

Eukaryotic cells are populated by membrane-enclosed organelles possessing discrete molecular and biochemical properties. Communication between organelles is established by shuttling vesicles that transport proteins and other molecules. Vesicles bud from a donor organelle, travel in the cytosol, and are delivered to a target organelle. All these steps are regulated to ensure that cargoes are transported in a specific and directed manner. The focus of this thesis is on the last part of the journey of a vesicle: the process of vesicle targeting. Two phases can be distinguished in this process: vesicle tethering, defined as the first interaction between the shuttling vesicle and the target membrane, and membrane fusion, which is the mixing of the lipid bilayers and of lumen content. Both phases are mediated by a minimal set of molecular components that include one member of the family of Rab GTPases, a vesicle tethering factor, a phosphoinositide lipid, and four SNAREs together with their regulatory proteins. While many studies have investigated the molecular details of how SNAREs mediate membrane fusion, the process of vesicle tethering is less well understood. The overall scope of my study is to describe the molecular details of vesicle tethering and how they can contribute to the general process of vesicle targeting. To address this question I developed an in vitro assay where I reconstitute in vitro the process of vesicle tethering. This bottom-up approach allows the molecular dissection of cellular processes outside of the complex context of the cell. With this assay I have characterized the vesicle tethering abilities of individual proteins involved in vesicle tethering on early endosomes. I show that a minimal vesicle tethering machinery can be formed by the concomitant interaction between one vesicle tethering factor and a phosphoinositide on the membrane of one vesicle, and by a vesicle tethering factor and a Rab GTPase on the membrane of another vesicle. These results provide an explanation for how vesicle tethering contributes to the specificity of vesicle targeting and to the directionality of cargo transport. In particular, specificity of vesicle targeting can arise from the specific interaction between a Rab and a vesicle tethering factor that is an effector of the Rab. I show that the asymmetric distribution of binding sites in the structure of a vesicle tethering factor can generate a heterotypic vesicle tethering reaction that can account for the directionality of cargo transport. The outcome of this thesis emphasizes the role that vesicle tethering factors have in the self-organized system of vesicle trafficking of eukaryotic cells. To identify novel Rab5 effectors implicated in vesicle tethering, I carried out a Rab5-chromatography on mouse liver. Amongst other novel Rab5 effectors, I identify a multi-subunit vesicle tethering complex that was not previously characterized in mammalian cells. The complex, named CORVET, is conserved from yeast to humans and plays a major role in cell physiology since its removal causes embryonic death in mice. I define its subunits composition, determine its subcellular localization, and elucidate its role in cargo transport. This finding reconciles a disharmony between findings in mammals and yeast regarding the molecular machinery responsible for the conversion from early to late endosomes. I also show that the newly identified subunit of the mammalian CORVET complex is the only Rab5 effector to localize to autophagosomes. I hypothesise that it is through the CORVET complex that Rab5 is involved in the formation and maturation of autophagosomes.

An estuarine mollusc Melanopsus trifasciata was studied with respect to its osmoregulatory capacity. Analysis of the environment, body fluids, and cell constituents was carried out and the results indicated that Melanopsus was able to regulate its cellular amino compounds in response to salinity changes. However it is isosmotic and more or loss isotonic with the environment as far as the haemocoelic fluid is concerned. An attempt was made to find out how or why this response of the cells to changing external osmotic pressure comes about. Various techniques were used: puromycin to block protein synthesis, labelled amino acids, time experiments, enzyme assays, and so on. Finally in the light of present day work the results were interpreted and a system of control put forward for Melanopsis which was later extended in general terms to all euryhaline poikilosmotic invertebrates.

An estuarine mollusc Melanopsus trifasciata was studied with respect to its osmoregulatory capacity. Analysis of the environment, body fluids, and cell constituents was carried out and the results indicated that Melanopsus was able to regulate its cellular amino compounds in response to salinity changes. However it is isosmotic and more or loss isotonic with the environment as far as the haemocoelic fluid is concerned. An attempt was made to find out how or why this response of the cells to changing external osmotic pressure comes about. Various techniques were used: puromycin to block protein synthesis, labelled amino acids, time experiments, enzyme assays, and so on. Finally in the light of present day work the results were interpreted and a system of control put forward for Melanopsis which was later extended in general terms to all euryhaline poikilosmotic invertebrates.

An estuarine mollusc Melanopsus trifasciata was studied with respect to its osmoregulatory capacity. Analysis of the environment, body fluids, and cell constituents was carried out and the results indicated that Melanopsus was able to regulate its cellular amino compounds in response to salinity changes. However it is isosmotic and more or loss isotonic with the environment as far as the haemocoelic fluid is concerned. An attempt was made to find out how or why this response of the cells to changing external osmotic pressure comes about. Various techniques were used: puromycin to block protein synthesis, labelled amino acids, time experiments, enzyme assays, and so on. Finally in the light of present day work the results were interpreted and a system of control put forward for Melanopsis which was later extended in general terms to all euryhaline poikilosmotic invertebrates.

An estuarine mollusc Melanopsus trifasciata was studied with respect to its osmoregulatory capacity. Analysis of the environment, body fluids, and cell constituents was carried out and the results indicated that Melanopsus was able to regulate its cellular amino compounds in response to salinity changes. However it is isosmotic and more or loss isotonic with the environment as far as the haemocoelic fluid is concerned. An attempt was made to find out how or why this response of the cells to changing external osmotic pressure comes about. Various techniques were used: puromycin to block protein synthesis, labelled amino acids, time experiments, enzyme assays, and so on. Finally in the light of present day work the results were interpreted and a system of control put forward for Melanopsis which was later extended in general terms to all euryhaline poikilosmotic invertebrates.

An estuarine mollusc Melanopsus trifasciata was studied with respect to its osmoregulatory capacity. Analysis of the environment, body fluids, and cell constituents was carried out and the results indicated that Melanopsus was able to regulate its cellular amino compounds in response to salinity changes. However it is isosmotic and more or loss isotonic with the environment as far as the haemocoelic fluid is concerned. An attempt was made to find out how or why this response of the cells to changing external osmotic pressure comes about. Various techniques were used: puromycin to block protein synthesis, labelled amino acids, time experiments, enzyme assays, and so on. Finally in the light of present day work the results were interpreted and a system of control put forward for Melanopsis which was later extended in general terms to all euryhaline poikilosmotic invertebrates.