The C Isoform of Dictyostelium Tetraspanins Localizes to the Contractile Vacuole and Contributes to Resistance against Osmotic Stress.
Tetraspanins (Tsps) are membrane proteins that are widely expressed in eukaryotic organisms. Only recently, Tsps have started to acquire relevance as potential new drug targets as they contribute, via protein-protein interactions, to numerous pathophysiological processes including infectious diseases and cancer. However, due to a high number of isoforms and functional redundancy, knowledge on specific functions of most Tsps is still scarce. We set out to characterize five previously annotated Tsps, TspA-E, from Dictyostelium discoideum, a model for studying proteins that have human orthologues. Using reverse transcriptase PCRs, we found mRNAs for TspA-E in the multicellular slug stage, whereas vegetative cells expressed only TspA, TspC and, to a lesser extent, TspD. We raised antibodies against TspA, TspC and TspD and detected endogenous TspA, as well as heterologously expressed TspA and TspC by Western blot. N-deglycosylation assays and mutational analyses showed glycosylation of TspA and TspC in vivo. GFP-tagged Tsps co-localized with the proton pump on the contractile vacuole network. Deletion strains of TspC and TspD exibited unaltered growth, adhesion, random motility and development. Yet, tspC- cells showed a defect in coping with hypo-osmotic stress, due to accumulation of contractile vacuoles, but heterologous expression of TspC rescued their phenotype. In conclusion, our data fill a gap in Dictyostelium research and open up the possibility that Tsps in contractile vacuoles of e.g. Trypanosoma may one day constitute a valuable drug target for treating sleeping sickness, one of the most threatening tropical diseases.
Department of Medicinal and Pharmaceutical Chemistry, Christian-Albrechts-University of Kiel, Kiel, Germany.
Tineke
T
Albers
Department of Cell Biology, University of Kassel, Kassel, Germany.
Markus
M
Maniak
Department of Medicinal and Pharmaceutical Chemistry, Christian-Albrechts-University of Kiel, Kiel, Germany.
Eric
E
Beitz
Department of Medicinal and Pharmaceutical Chemistry, Christian-Albrechts-University of Kiel, Kiel, Germany.
Julia
J
von Bülow
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000302:isolation & purification; D015153:Blotting, Western; D004023:Dictyostelium / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009997:Osmotic Pressure; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017494:Proton Pumps / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D059470:Tetraspanins / Q000235:genetics* / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism* / Q000648:ultrastructure
D000906:Antibodies; D020033:Protein Isoforms; D017494:Proton Pumps; D015800:Protozoan Proteins; D012333:RNA, Messenger; D059470:Tetraspanins; D049452:Green Fluorescent Proteins