Untersuchungen zur Expression und subzellulären Lokalisation der v-SNARE Isoformen VAMP-1a und -1b

Die sogenannten SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) - Proteine sind essentielle Bestandteile der zellulären Transportmaschinerie und maßgeblich beteiligt an Membranfusionsprozessen, wie z.B. der Fusion zwischen einem Transportvesikel- und seiner Zielmembran....

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Bibliographic Details
Main Author: Rodepeter, Fiona Rosali
Contributors: Wiegand, Susanne (Prof. Dr. med.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2014
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The so-called SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are essential constituents of the intracellular trafficking machinery and are primarily involved in membrane fusion processes such as fusion of a transport vesicle with its cognate target membrane. The v-SNARE protein VAMP-1 (vesicle associated membrane protein 1) that is found in transport vesicle membranes is a so-called type II membrane protein consisting of a dominant N-terminal cytosolic, a transmembrane and a short intraluminal C-terminal domain. The mechanism of alternative RNA-splicing that contributes to the existence of different VAMP isoforms could also be found in mammalian cells. This mechanism was demonstrated for the first time for the VAMP-1 gene of the rat describing two differentially expressed VAMP-1 isoforms termed VAMP-1a and VAMP-1b. The newly identified VAMP-1b isoform differed from the known VAMP-1a isoform in its C-terminal intraluminal end and was mainly expressed in nonneuronal particularly secretory active tissues such as the parotid gland, whereas the known VAMP-1a isoform exhibited a primary distribution in the central nervous system. In addition, it was reported that a similar human VAMP-1 splice isoform exhibiting a variable intraluminal C-terminal end was sorted to mitochondria, pointing to the C-terminal end of these SNARE proteins to potentially act as sorting signals. The aim of the present study was to evaluate if both VAMP-1 isoforms differ in their subcellular localization by using a cell biological approach involving fluorescence microscopy since this could point to a role of the intraluminal C-terminal end for sorting of both isoforms to different subcellular compartments. To approach these questions GFP and RFP labelled VAMP-1a (GFP- and RFP-V1a) and VAMP-1b (GFP- and RFP-V1b) expression constructs were generated by PCR cloning and were cotransfected into HeLa cells with subsequent evaluation of the protein’s subcellular localization using fluorescence microscopy. Regions consistent with the endoplasmic reticulum (ER) and Golgi apparatus demonstrated a clear overlap of both signals. Since VAMP proteins are integrated into membranes of the ER after synthesis, and differences in the localization are expected to occur after sorting in the trans-Golgi network (TGN), this was an expected finding, in particular since VAMP-1 should play a role in the late secretory pathway. In the periphery of the cells, vesicular structures could be observed which seemed to express only one of both isoforms when coexpressed (GFP-V1a/RFP-V1b or GFP-V1b/RFP-V1a). To evaluate colocalization of both proteins in non-fixed vital cells, the method of total internal reflection fluorescence microscopy (TIRF) was implemented. For this, the respective constructs were expressed in COS-7 cells, since these were particularly suitable for TIRF microscopy, demonstrating good expression of the fluorescent proteins. Similarly as observed in HeLa cells, vesicular structures could also be seen that primarily expressed one of both isoforms. In addition, evidence for homotypic fusion events was found between vesicles that coexpressed VAMP-1a as well as VAMP-1b. To evaluate a possible localization of the two isoforms in compartments of the late secretory pathway, GFP-V1a or GFP-V1b were transfected into HeLa cells and subsequent immunocytochemistry was performed, using antibodies directed against marker proteins specific for mitochondria (TOM20), early endosomes (EEA1), lysosomes (LAMP-3) and autophagosomes (MAP LC3beta), as well as coexpression of RFP-V1a or RFP-V1b with the peroxisomal marker protein GFP-SKL. Taken together, no evidence was found for sorting of both VAMP-1 isoforms to mitochondria or peroxisomes. However, both isoforms were found to be expressed in a minor subset of singular vesicles that appeared associated with the early endosomal, lysosomal and autophagosomal compartment. Since vesicular structures were observed that expressed only one of the two transfected VAMP-1 isoforms, it is possible that only one of both isoforms is sorted to a yet unidentified compartment. Subsequent studies, using other methods such as subcellular fractionation, could help to further define these compartments.