The glucose transporter 4 (GLUT4) is the major insulin-responsive glucose transporter in adipose and muscle tissues. Although the early steps in the insulin signaling pathway governing translocation of GLUT4 to the plasma membrane are well understood, the final steps in this pathway are not. TUG is a protein which has been shown to affect trafficking of GLUT4 both in the basal state and in response to insulin. One protein-protein interaction between TUG and the large cytosolic loop of GLUT4 has previously been identified. Based on reports of the requirement of the GLUT4 N-terminal domain for its proper targeting to the plasma membrane, we postulated that an interaction might also exist between TUG and the N-terminal domain of GLUT4, and we tested this hypothesis using two sets of pull-down experiments. In the first set, using the N-terminal domain of GLUT4 fused with glutathione S-transferase (GST), we were able to pull TUG down from the lysates of TUG-transfected HEK 293 cells. TUG was also pulled down by the GLUT4 cytosolic loop and, to a much lesser extent, its C-terminal domain. However, there was no specific interaction between these fusion proteins and the lysates of cells transfected with a truncated form of TUG lacking its own N-terminal domain. In the second set of experiments, using a biotinylated synthetic GLUT4 N-terminal peptide, we pulled down a protein detected by an anti-TUG antibody and running at ~64 kDa, a slightly higher molecular weight than wild-type TUG. We believe that this band represents modified full-length TUG. This interaction was not seen using synthetic GLUT4 N-terminal peptide mutated at 4 amino acids previously identified as necessary for proper GLUT4 retention and insulin-responsive trafficking. We conclude that TUG interacts not only with the large cytosolic loop of GLUT4, but also with the N-terminal domain of GLUT4, and that this latter interaction can be disrupted by mutations in GLUT4 that cause defective trafficking, suggesting that this interaction is critical for GLUT4 intracellular retention and insulin-responsive GLUT4 trafficking.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 12, 2009). Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 11, 2009). Includes bibliographical references.

Abstract In mammalian cells the pH gradient between the organelles, cytoplasm and extracellular space is strictly regulated. Maintenance of pH homeostasis is crucial for the normal function of the cell and its organelles. In solid tumours, cells often suffer from hypoxia, deprivation of nutrients and acidic extracellular milieu as a result of inadequate vascularisation. Cancer cells are also known to suffer from other pH abnormalities. Defective acidification of intracellular organelles as well as a reversed pH gradient across the plasma membrane have been detected in numerous tumour tissues and cells. Aberrant secretion of lysosomal hydrolases, loss of cell polarity and increased expression of tumour-specific proteins are common phenotypic changes of cancer cells. In this study, secretion of cathepsin D, a lysosomal aspartic hydrolase, was shown to result from the acidification defect of cancer cells. In normal cells cathepsin D is sorted in the Golgi complex by mannose-6-phosphate receptors and transported via endosomes to lysosomes. In breast and colorectal cancer cells having abnormally neutral endosomes receptors were shown to accumulate in endosomes resulting in the aberrant secretion of newly synthesised cathepsin D from the cells. Carcinoembryonic antigen (CEA) is an oncofetal protein widely used as a tumour follow-up marker. It is normally expressed at low levels and is localised at the apical surface of epithelial cells via a glycosyl phosphatidyl inositol (GPI) anchor. In cancer cells the expression of CEA is increased and the protein is found over the entire surface of cells. In this study, the tumour microenvironmental factors, hypoxia and abnormal pH homeostasis, were shown to increase the expression of carcinoembryonic antigen in cancer cells. In addition, the absence of acidic organelles was shown to induce mistargeting of CEA to the basolateral membrane in polarised cells. The abnormally neutral Golgi was found to interfere with the complex formation of carcinoembryonic antigen, a phenomenon recently associated with the apical sorting of other GPI-anchored proteins. Altogether these results emphasise the role of tumour-related factors – altered pH homeostasis and hypoxia – in the phenotypic changes of cancer cells.

No description available.

Mitochondrion is believed to be an ubiquitous organelle which occurred about 1,5 billion years ago by a single endosymbiotic event. Mitochondria is mostly dependent on the protein import from cytosol thus the establishment of protein import machinery was essential for seizing the new endosymbiont. Possibilities of studying the evolution of protein import machineries are quite limited given that no "free living" mitochondria or amitochondriate organisms are known nowadays. One alternative is to study mitochondrial secondary reductive evolution of anaerobic parasitic protists. Giardia intestinalis is flagellated protozoan living in microaerofilic environment of the small intestine. It containes one of the most reduced mitochondrion (mitosome) described so far. Hence it serves as a great model for studying mitochondrial evolution. Although it is well understood that all mitosomal proteins are transported from cytosol, many aspects of protein import pathway remain elusive. While the main channel Tom40 is present in the outer membrane, two other main translocases (Sam50 which is required for betta-barrel assembly in the outer membrane and Tim17/22/23 which is essential for protein translocation through the inner membrane) have not been identified so far. Protein translocation through Tim17/22/23 channel...

The majority of the mitochondrial proteins is synthetized on the cytosolic ribosomes in the form of the protein precursors bearing mitochondrion-targeting signal presequences. Once the protein precursor has reached the mitochondrial matrix the signal presequence is no longer necessary and is cleaved off by heterodimeric mitochondrial processing peptidase (MPP; α/β). Although the crystal structure of MPP is available, the MPP mechanism of function is still matter of discussion. An all atomic, non-restrained molecular dynamics (MD) simulation in explicit water was used to study in detail the structural features of the highly conserved glycine-rich loop (GRL) of the regulatory α-subunit of the yeast MPP. Wild-type and GRL-deleted MPP structures were studied both in the presence and absence of a substrate in the peptidase active site. Targeted MD simulations were employed to study the mechanism of substrate translocation from the GRL to the peptidase active site. We demonstrate that the natural conformational flexibility of the GRL is crucial for the substrate translocation process from outside the enzyme towards the MPP active site. We show that the α-helical conformation of the substrate is important not only during its initial interaction with MPP (i.e. substrate recognition), but also later, at...

Sistemas de transporte tipo ABC são responsáveis pelo transporte de uma grande variedade de substâncias dentre elas os oligopeptídeos e molibdato. Neste trabalho estudamos dois sistemas de transportadores do tipo ABC (mod, envolvido na captação de molibdato e o opp na capação de oligopeptídeos) presentes na bactéria Xanthomonas axonopodis pv. citri (Xac). Em particular analisamos a organização genética dos óperons mod e as proteínas ModA e OppA, componentes solúveis localizados no periplasma e responsáveis pela ligação aos substratos. Por meio de técnicas de modelagem molecular, definimos modelos estruturais para as proteínas ModA e OppA. Para a proteína ModA caracterizamos cinco resíduos que compõem a cavidade ligadora e são responsáveis pelas interações com o íon molibdato, assim como a sua similaridade estrutural e sequencial com ortólogos de 3 grupos distintos de bactérias. Para a OppA, descrevemos o seu comportamento na ancoragem de diferentes oligopeptídeos. Avaliamos parâmetros como a extensão da cadeia e estabelecemos uma ordem crescente de afinidade entre os oligopeptídeos com diferente composição residual e a proteína OppA. / ABC transport system are responsable for the uptake of a large variety of substrates, including oligopeptides and molybdate. In this work we studied two ABC transporter systems (mod and opp responsable for molybdate and oligopeptide uptake, respectively) present in plant pathogen Xanthomonas axonopodis pv. citri (Xac). We investigated the genetic organization of mod operon and, particularly, structural feature of periplasmic components, ModA and OppA proteins, of the uptake systems. Using molecular modeling techniques, we defined the structural models of both ModA and OppA proteins. Based on the ModA structural model, amino acid residues involved in molybdate interaction were identified. In addition, both the structural and sequence similarities of Xac ModA and other bacterial orthologs with experimentally defined structural organizations were described. Based on the OppA structural model, we applied molecular docking tools to determine the binding specificity for different oligopeptide regarding number and amino acid composition. Collectively, our results represent an important contribution to the study of ABC transporters in an economically relevant phytopathogen bacterial species.

Brefeldin A (BFA) has been a useful tool for studying organelle dynamics and protein trafficking in plant cells. Using several Golgi (MAN1 and GONST1) and TGN (SCAMP1 and SYP61) fluorescent protein markers as tools, I have showed that BFA-induced aggregates from Golgi apparatus and TGN are morphologically distinct in the same plant cells. In addition, the internalized endosomal marker FM4-64 colocalized with the TGN-derived aggregates but separated from the Golgi aggregates. In the presence of the endocytosis inhibitor tyrphostin A23, SCAMP1 and FM4-64 are largely excluded from the TGN SYP61-positive BFA-induced aggregates, indicating homotypic fusion of TGN rather than de novo endocytic trafficking is important for the formation of TGN/EE-derived BFA-induced aggregates. Since the TGN also serves as an EE receiving materials from plasma membrane continuously, these data therefore support the notion that the secretory Golgi organelle is distinct from the endocytic TGN/EE in response to BFA treatment in plant cells. / Little is known about the trafficking mechanism of plasma membrane (PM) proteins in the endomembrane system of plant cells that contain several membrane-bound organelles including the endoplasmic reticulum (ER), Golgi, trans-Golgi network (TGN) of early endosome (EE), prevacuolar compartment (PVC) or late endosome (LE). Here, I study the transport pathway and sorting signals of secretory carrier membrane protein 1 (SCAMP1) by following its transient expression in tobacco BY-2 protoplasts and show that SCAMP1 reaches the PM via an ER-Golgi-TGN-PM pathway. Loss-of-function and gain-of-function analysis of various GFP fusions with SCAMP1 mutations further demonstrates that: (1) the cytosolic N terminus of SCAMP1 contains an ER export signal; (2) the transmembrane domain 2 (TMD2) and TMD3 of SCAMP1 are essential for Golgi export; and (3) SCAMP1 TMD1 is essential for TGN-to-PM targeting. Therefore, both the cytosolic N-terminus and TMD sequences of SCAMP1 play integral roles in mediating its transport to the PM via an ER-Golgi-TGN pathway. / Cai, Yi. / Adviser: Liwen Jiang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 93-102). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (Ph.D.)--University of Central Florida, 2008. / Adviser: Sic L. Chan. Includes bibliographical references (p. 74-84).