No description available.

No description available.

Constitutive secretion is responsible for the targeting of transmembrane proteins to the plasma membrane, and for the secretion of extracellular matrix proteins, hormones, and other cellular products. The basic steps of secretion are well understood – proteins synthesized in the endoplasmic reticulum are transported in lipid-bound intermediates to the Golgi, and from the Golgi to the plasma membrane or cell exterior. Dysfunction of the secretory pathway – either constitutive or regulated – is involved in many disease states. One such state is diabetic nephropathy (DN). DN is characterized by renal hypertrophy and fibrosis, and is the leading cause of renal failure worldwide. Our lab had previously shown that munc13 is both upregulated and activated in the diabetic kidney, and that munc13 is an effector of rab34. Study of rab34 in HeLa cells revealed that rab34 is localized to the Golgi, and that it is required for the secretion of the Vesicular Stomatitis Virus glycoprotein. Colocalization experiments, as well as the use of Brefeldin A, localized the effect of rab34 to intra-Golgi transport. Further experiments indicated that glucose-induced upregulation of munc13 in rat mesangial cells increased the rate of constitutive secretion to the plasma membrane, and that this effect depended on its interaction with rab34. Finally, munc13 and rab34 were found to be required for the high glucose-mediated stimulation of Transforming Growth Factor-β secretion from mesangial cells, placing these two proteins at a key point in a pathway of physiological significance in the pathology of DN.

Constitutive secretion is responsible for the targeting of transmembrane proteins to the plasma membrane, and for the secretion of extracellular matrix proteins, hormones, and other cellular products. The basic steps of secretion are well understood – proteins synthesized in the endoplasmic reticulum are transported in lipid-bound intermediates to the Golgi, and from the Golgi to the plasma membrane or cell exterior. Dysfunction of the secretory pathway – either constitutive or regulated – is involved in many disease states. One such state is diabetic nephropathy (DN). DN is characterized by renal hypertrophy and fibrosis, and is the leading cause of renal failure worldwide. Our lab had previously shown that munc13 is both upregulated and activated in the diabetic kidney, and that munc13 is an effector of rab34. Study of rab34 in HeLa cells revealed that rab34 is localized to the Golgi, and that it is required for the secretion of the Vesicular Stomatitis Virus glycoprotein. Colocalization experiments, as well as the use of Brefeldin A, localized the effect of rab34 to intra-Golgi transport. Further experiments indicated that glucose-induced upregulation of munc13 in rat mesangial cells increased the rate of constitutive secretion to the plasma membrane, and that this effect depended on its interaction with rab34. Finally, munc13 and rab34 were found to be required for the high glucose-mediated stimulation of Transforming Growth Factor-β secretion from mesangial cells, placing these two proteins at a key point in a pathway of physiological significance in the pathology of DN.

No description available.

The secretory pathway of plant cells consists of several organelles that are connected by vesicle and tubular transport. Every compartment has a distinct function and the specificity of vesicle fusion is essential to maintain the organelles identity. N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a crucial role in the secretory pathway driving specific vesicle fusions. A vesicle SNARE (v-SNARE) on a vesicle specifically interacts with two or three target SNAREs (t-SNAREs) on the target compartment. This event leads to vesicle membrane fusion with the membrane of the target compartment and the release of cargo molecules into the organelle lumen.<p> The aim of this work was the characterization of two <i>Arabidopsis thaliana</i> SNAREs. The first one is a v-SNARE, Bet11 that is the Arabidopsis ortholog of the yeast and mammal ER-Golgi v-SNARE, Bet1. In these organisms, Bet1 is involved in trafficking between the ER and Golgi apparatus. The second protein studied is a putative SNARE called Bet12 that shares high sequence identity with Bet11. In particular, I was interested in studying the sorting of these two proteins and their role in the secretory pathway of plant cells. By confocal laser microscopy, I demonstrated that these two proteins have different intracellular localization: Bet11 was mainly localized on the ER, Golgi stacks and punctate structures that I have identified as endosomes. Bet12 was localized only on the Golgi stacks. The identification of signal(s) involved in targeting of Bet11 and Bet12 were studied. To reach this aim I generated different mutant chimeras of Bet11 and Bet12. The co-expression of these chimeras with specific protein markers suggested that the distribution of these proteins was the result of a combined influence of multiple domains.<p> A serine in the Bet11 sequence was identified as a putative phosphorylation site and appeared important for proper Bet11 intracellular distribution.<p> The different intracellular distributions of Bet11 and Bet12 suggest different biological roles for the two proteins. To functionally characterize these two proteins homozygous knock-down mutants of Bet11 were screened. These plants had no evident phenotype, suggesting a possible genetic redundancy in this SNARE family.

The secretory pathway of plant cells consists of several organelles that are connected by vesicle and tubular transport. Every compartment has a distinct function and the specificity of vesicle fusion is essential to maintain the organelles identity. N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a crucial role in the secretory pathway driving specific vesicle fusions. A vesicle SNARE (v-SNARE) on a vesicle specifically interacts with two or three target SNAREs (t-SNAREs) on the target compartment. This event leads to vesicle membrane fusion with the membrane of the target compartment and the release of cargo molecules into the organelle lumen.<p> The aim of this work was the characterization of two <i>Arabidopsis thaliana</i> SNAREs. The first one is a v-SNARE, Bet11 that is the Arabidopsis ortholog of the yeast and mammal ER-Golgi v-SNARE, Bet1. In these organisms, Bet1 is involved in trafficking between the ER and Golgi apparatus. The second protein studied is a putative SNARE called Bet12 that shares high sequence identity with Bet11. In particular, I was interested in studying the sorting of these two proteins and their role in the secretory pathway of plant cells. By confocal laser microscopy, I demonstrated that these two proteins have different intracellular localization: Bet11 was mainly localized on the ER, Golgi stacks and punctate structures that I have identified as endosomes. Bet12 was localized only on the Golgi stacks. The identification of signal(s) involved in targeting of Bet11 and Bet12 were studied. To reach this aim I generated different mutant chimeras of Bet11 and Bet12. The co-expression of these chimeras with specific protein markers suggested that the distribution of these proteins was the result of a combined influence of multiple domains.<p> A serine in the Bet11 sequence was identified as a putative phosphorylation site and appeared important for proper Bet11 intracellular distribution.<p> The different intracellular distributions of Bet11 and Bet12 suggest different biological roles for the two proteins. To functionally characterize these two proteins homozygous knock-down mutants of Bet11 were screened. These plants had no evident phenotype, suggesting a possible genetic redundancy in this SNARE family.

The activation of many pro-hormones occurs through cleavage at pairs of basic residues and is mediated by two serine proteases, PC2 and PC3. Like their substrates, they are also synthesised as inactive precurors (pro-PC2 and pro-PC3). Maturation is autocatalytic and requires removal of the N-terminal pro-peptide. Pro-PC3 matures within the endoplasmic reticulum (ER), whereas maturation of pro-PC2 proceeds within the later compartments of the Golgi network (TGN)/secretory vesicle (SV) and is thought to be regulated by 7B2. In this study the molecular basis for the differences in the maturation location and the interaction with 7B2 was examined by performing domain swap and site directed mutagenesis experiments. The mutant constructs were analysed within an <I>in vitro</I> cell free system. The results suggest that the oxyanion hole residue (Asp³¹⁰) of pro-PC2 restricts maturation to a late secretory compartment and is important for the interaction with 7B2. Mutation of this residue to resemble that of PC3 (Asp310Ans), altered pro-PC2 maturation from a TGN/SV like environment to an ER like environment. Maturation of pro-PC2, but not pro-PC3, was shown to be inhibited by 7B2. Residue Asp³¹⁰ of PC2 was necessary to mediate this interaction. When a similar mutant of PC3 was created to resemble PC2 (Asn309Asp), this residue was not sufficient to alter the maturation profile of pro-PC3 nor was it able to confer 7B2 sensitivity. The pro-region of PC2 was sufficient to alter maturation of PC3 from the ER-like compartment to a TGN/SV-like compartment, but was not able to confer 7B2 sensitivity onto PC3. This study also demonstrated that a basic cluster (HHKQQ⁸⁸) of pro-PC2 was important for delaying maturation to a late compartment and that the residue Phe¹⁰⁴, was important for efficient maturation. Mutational analysis of pro-7B2 revealed that a region within residues 1-151 was important for the interaction with pro-PC2.

No description available.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Sept. 9, 2009). Includes bibliographical references.