Forschungsgruppe Anne Spang
Intracellular transport in yeast and worms
Asymmetry is an inherent property of most cells. Proteins and mRNA have to be distributed at specific cellular locales to perform their proper function or to be translated in a spatially and temporally regulated manner. Although the localization of the mRNAs is restricted to the cytoplasmic face of intracellular organelles or the plasma membrane, proteins and lipids have to be localized to these organelles to provide a platform on which mRNAs and/or proteins can be recruited and restricted. In general this compartmentalization is achieved by intracellular transport through exocytic (secretory pathway) and endocytic avenues. Communication between different organelles is maintained in large part by transport vesicles that are covered with a proteinaceous coat, which polymerizes and which helps to recruit cargo proteins into the nascent transport vesicle. One class of small GTPases - the family of Arf and Sar GTPases - is essential for the generation of transport carriers, while another class - Rab GTPases - is involved in the consumption of transport carriers and seems to play an essential role in the maintenance of organellar identity.
Our research interests center around questions like how intracellular traffic contributes to cellular asymmetry and how intracellular processes are regulated by small GTPases of the Arf and Rab families. We use the unicellular yeast Saccharomyces cerevisiae and the roundworm Caenorhabditis elegans for our studies as both organisms are particularly well suited to answer the kind of questions we like to address.
The regulation of Arf family proteins
In recent times, we have investigated the role of GTPase activating proteins for Arf1p. We could show that the yeast homologues of ArfGAP1 and ArfGAP2/3, Gcs1p and Glo3p have overlapping functions in retrograde transport from the Golgi apparatus to the ER (Poon et al., 1999), and that Glo3p is an integral part of the COPI coat, which mediates this transport step (Lewis et al., 2004). The finding that ArfGAPs can induce a conformational change in SNARE proteins, which are essential components in membrane fusion processes (Rein et al., 2002, Robinson et al., 2006, Schindler and Spang, 2007), prompted us to investigate more closely the role of the ArfGAP2/3 Glo3p in transport vesicle formation. We identified a region in Glo3p, which binds to SNAREs, coatomer and cargo (BoCCS) (Schindler et al., 2009). Moreover, the C-terminal Glo3 regulatory motif, GRM appears to transmit the Arf1p nucleotide state via the GAP domain to the BoCCS region. Upon stimulation of the GTPase activity, SNAREs, coatomer and cargo could be released from the BoCCS region. We are currently trying to understand the molecular rearrangements in Glo3p and to identify interaction partners to gain further insights in the regulation of Glo3p. We also returned recently again to the analysis of the function of different Arf guanine nucleotide exchange factors (ArfGEFs) (Spang et al., 2001) and investigate their roles in Caenorhabditis elegans.
The regulation of cargo sorting and transport
In our quest to understand the life cycle of a transport vesicle, we realized that cargo, which needs to be transported in vesicles, is not just a passive bystander, but plays a more active role. Overexpression of cargo proteins with a coatomer-binding sequence (-KKXX) can rescue coatomer mutants in the –KKXX recognizing subunit (Sandmann et al., 2003). Furthermore, in the absence of the ArfGAP Glo3p, the p24 family proteins, which cycle between the ER and the Golgi apparatus, are required to bud efficiently vesicles from the Golgi (Aguilera et al., 2008). Moreover, in collaboration with Blanche Schwappach, we identified a novel bi-partite cargo recognition motif in coatomer (Michelsen et al., 2007). These results strongly indicate that cargo-coat interaction stabilize the priming complexes suggested by Springer et al. (1999) and that the formation of coat-cargo complexes is an essential integral part of vesicle biogenesis. We also demonstrated that Ypt1p is the Rab-GTPase responsible for anterograde and retrograde transport in the ER-Golgi shuttle as well as for Golgi maintenance in S. cerevisiae (Kamena et al., 2008). Finally, we have identified a novel trans-Golgi localized complex, exomer, which is required for the sorting and transport of specific cargo to the plasma membrane (Trautwein et al., 2006, Zanolari et al., 2010, Rockenbauch et. al., 2012). We have found more cargo proteins that follow this pathway and are in the process of investigating the cargo-exomer interaction interface and decipher the transport mechanisms.
The regulation of early-to-late endosomal transport
Recently, we cloned a C. elegans mutant, sand-1(or552) that shows a defect in endocytosis. While initial uptake of material was normal in oocytes and coelomocytes, the transport from early-to-late endosomes seemed to be blocked (Poteryaev and Spang, 2005; Poteryaev et al., 2007). sand-1(or552) mutants had strongly enlarged early endosomes, which were positive for the small GTPase RAB-5. In contrast, RAB-7, the Rab protein normally found on late endosomes was mislocalized to the cytoplasm. This finding opened the possibility that SAND-1 was a regulator of early-to-late endosome transition. We followed up on this hypothesis and could show that in coelomocytes early-to-late endosome transport is performed through Rab conversion, and not through vesicle transport. We went on to demonstrate that SAND-1 actively interrupts the activation of RAB-5 by displacing the guanine nucleotide exchange factor of RAB-5, RABX-5 from early endosomes (Poteryaev et al., 2010). At the same time SAND-1 helps to recruit RAB-7 to endosomes to drive Rab conversion, indicating that SAND-1 acts as a critical switch in endosome maturation. These functions of SAND-1 are also conserved in mammalian cells (Poteryaev et al., 2010). We are now investigating the regulation of SAND-1 function and how multi-vesicular body formation, recycling pathways and endosome maturation are coordinated.
The regulation of mRNA metabolism and transport
This research direction was inspired by our finding that the poly A binding protein, Pab1p, associates with Arf1p and COPI vesicles in an mRNA-dependent manner and that Arf1p is required for ASH1 mRNA localization to the bud tip of yeast cells (Trautwein et al., 2004). The subsequent analysis allowed us to identify the first distal pole-localized mRNA in yeast (Kilchert and Spang, 2011) and to identify a novel pathway by which mRNAs are sequestered in processing bodies (P-bodies) for their degradation (Kilchert et al., 2010). We performed screens to identify mRNAs that are restricted to certain sites and are currently investigating the mechanism of the localization.