Download Intracellular trafficking and mis-trafficking of disease

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.