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Project 2 (Molecular & Cell Biology, Saarland University) Intracellular trafficking and mis-trafficking of disease-related plasma membrane proteins in yeast and mammalian cells Correct subcellular protein localization is not only essential for normal protein function, it likewise determines proper access to individual interacting partners and specific posttranslational modifications. Consequently, aberrantly localized proteins have been linked to a wide range of human diseases including Alzheimer, cancer and even kidney stones (1). In the current PhD project, expression and subcellular targeting of two types of plasma membrane proteins will be investigated in both, yeast and mammalian cells: (i) Wild-type and clinically relevant mutant variants of human anion exchanger AE1, and (ii) KDEL receptors (KDELR) and their compartmental distribution between the Golgi, the endoplasmic reticulum (ER) and the plasma membrane. Altered trafficking of the anion exchange transporter 1 (AE1) can cause a genetically inherited disease - distal renal tubular acidosis (dRTA) - which can be linked to various mutations within the chromosomal SLC4A1 gene (2). Normally, AE1 is expressed at the basolateral membrane of type A intercalated cells (kAE1) in the renal collecting duct where it mediates electroneutral chloride/bicarbonate exchange and participates in the fine-tuning of acid-base homeostasis. While dominant dRTA mutants retain wild-type kAE1 intracellularly, recessive dRTA mutants often show mistrafficking that in some cases can be rescued by coexpressing WT kAE1 (3). To mechanistically dissect the underlying mechanisms and principles for proper kAE1 targeting and mistrafficking – which is still largely unknown - the present PhD project will analyse kAE1 expression and trafficking in wild-type yeast cells and in various yeast knock-out mutants with defined defects in intracellular protein transport (4, 5). In case of yeast and mammalian KDEL receptors (KDELR) their essential cell function is to retrieve resident ER proteins like the chaperones BiP or protein disulfide isomerase PDI that escaped from the secretory pathway back to the ER (6). Interestingly, some clinically relevant A/B protein toxins such as cholera toxin, Pseudomonas exotoxin A or even the yeast K28 virus toxin possess a KDEL-like amino acid motif at the C-terminus of their cell binding B-subunits which is recognized and bound by KDELRs of their target cells (7, 8). Until now it was believed that the initial toxin interaction with KDELRs occurs within the Golgi, i.e. after receptor-mediated endocytosis and endosomal trafficking. However, we recently showed that yeast and mammalian KDELRs are not only present in the membranes of ER and Golgi but also at the cell surface where they can bind KDEL cargo at the plasma membrane and mediate cargo transport back to the ER via clathrin-mediated KDELR endocytosis and COPImediated retrograde transport. Within the PhD project, various aspects of KDELR and kAE1 targeting and mistargeting will be addressed at the molecular level by using cell biological and biochemical techniques, live-cell imaging and TIRF microscopy in collaboration with groups of the IRTG consortium. Reference List 1. Hung MC, Link W (2011). Protein localization in disease and therapy. J Cell Sci 124, 3381–3392. 2. Cordat E, Kittanakom S, Yenchitsomanus PT, Li J, Du K, et al. (2006). Dominant and recessive distal renal tubular acidosis mutations of kidney anion exchanger 1 induce distinct trafficking defects in MDCK cells. Traffic 7, 117–128. 3. Chu CYS, King JC, Berrini M, Alexander RT, Cordat E (2013). Functional rescue of a kidney anion exchanger 1 trafficking mutant in renal epithelial cells. PLoS ONE 8(2), e57062. 4. Zhao R, Reithmeier RAF (2001). Expression and characterization of the anion transporter homologue YNL275w in Saccharomyces cerevisiae. Am J Physiol Cell Physiol 281, C33–C45. 5. Carroll SY, Stirling PC, Stimpson HEM, Gießelmann E, Schmitt MJ, Drubin DG (2009). A yeast killer toxin screen provides insights into A/B toxin entry, trafficking, and killing mechanisms. Dev Cell 17, 552-560. 6. Capitani M, Sallese M (2009): The KDEL receptor: new functions for an old protein. FEBS Lett 583, 3863-3871. 7. Eisfeld K, Riffer F, Mentges J, Schmitt MJ (2000). Endocytotic uptake and retrograde transport of a virally encoded killer toxin in yeast. Mol Microbiol 37, 926-940. 8. Schmitt MJ, Breinig F (2006). Yeast viral killer toxins: lethality and self-protection. Nat Rev Microbiol 4, 212-221.