Worm Breeder's Gazette 16(2): 40
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
| 1 | Department of Pharmacology, Biocenter of the University of Basel, Klingelbergstr. 70, 4056 Basel, Switzerland |
| 2 | Department of Biology, F. Hoffmann-La Roche Ltd., Pharmaceutical Research-New Technologies, Basel, Switzerland |
Trafficking along the exocytic pathway involves a complex system of transport vesicles and membrane structures. The early secretory pathway includes the ER, the ER-Golgi intermediate compartment (ERGIC) and the Golgi apparatus that are connected by a vesicle-mediated anterograde (from the ER) and retrograde (to the ER) protein transport pathway. The ERGIC is now best defined by the marker protein ERGIC-53, a type I transmembrane protein.
In vitro and cell culture experiments revealed that ERGIC-53 is a calcium dependent mannose lectin continuously recycling in the early secretory pathway suggesting that it may operate as a transport receptor for a certain subset of glycoproteins (Vollenweider et al., 1998, Appenzeller et al., 1999).
Mutations in ERGIC 53 can lead to combined factor V/VIII deficiency in humans presumably due to inefficient secretion of these coagulation factors (Nichols et al., 1998). However the exact involvement of ERGIC-53 in the downregulation of these coagulation factors remains unclear.
ERGIC-53 is highly conserved through all the fila ranging from C. elegans to humans. In order to get a deeper insight into the in vivo function of ERGIC-53 we decided to use C. elegans as a model system. ERGIC-53 has only one othologue in C. elegans: K07A1.8.
The expression pattern was established using specific antisera. C.e.ERGIC-53 is ubiquitously expressed; however a stronger expression was observed in secretory tissues like: the gland cells of the pharynx, the spermatheca , the seminal vesicle and the vas deferens in males.
RNAi experiments with C.e.ERGIC-53 suggested a weak spe phenotype. We are now trying to generate deletion alleles.
Since ERGIC-53 has been shown in cell culture experiments to act as a cargo receptor for a subset of glycoproteins. It is our goal to find out which proteins interact with C.e.ERGIC-53. For this purpose we chemically crosslinked worm homogenates with the membrane permeable, reducible crosslinker DSP followed by immunoprecipitation using our antisera and SDS PAGE. Several candidates have already been detected by silver staining. We are now in the process of determine these proteins by MASSPEC and direct microsequencing.
The combination of reverse genetics and biochemistry will allow us to approach C.e.ERGIC-53 and its function from two sides. In addition we are investigating the only other member of this lectin family: T04G9.3. Preliminary datas provided by promotor::gfp constructs indicate a similar expression pattern as C.e.ERGIC-53.
Literature:
Vollenweider F., Kappeler F., Itin C., and Hauri HP. (1998). Mistargeting of the lectin ERGIC-53 to the endoplasmic reticulum of HeLa cells impairs the secretion of a lysosomal enzyme. J Cell Biol 142, 377-89.
Nichols WC., Seligsohn U., Zivelin A., Terry VH., Hertel CE., Wheatley MA., Moussalli MJ., Hauri HP., Ciavarella N., Kaufman RJ., and Ginsburg D. (1998). Mutations in the ER-Golgi intermediate compartment protein ERGIC-53 cause combined deficiency of coagulation factors V and VIII. Cell 93, 61-70.
Appenzeller C., Andersson H., Kappeler F., and Hauri HP. (1999). The lectin ERGIC-53 is a cargo transport receptor for glycoproteins. Nature Cell Biology 1, 330-334.