The C. elegans TspanC8 tetraspanin TSP-14 exhibits isoform-specific localization and function

Tetraspanin proteins are a unique family of highly conserved four-pass transmembrane proteins in metazoans. While much is known about their biochemical properties, the in vivo functions and distribution patterns of different tetraspanin proteins are less understood. Previous studies have shown that two paralogous tetraspanins that belong to the TspanC8 subfamily, TSP-12 and TSP-14, function redundantly to promote both Notch signaling and bone morphogenetic protein (BMP) signaling in C. elegans. TSP-14 has two isoforms, TSP-14A and TSP-14B, where TSP-14B has an additional 24 amino acids at its N-terminus compared to TSP-14A. By generating isoform specific knock-ins and knock-outs using CRISPR, we found that TSP-14A and TSP-14B share distinct as well as overlapping expression patterns and functions. While TSP-14A functions redundantly with TSP-12 to regulate body size and embryonic and vulva development, TSP-14B primarily functions redundantly with TSP-12 to regulate postembryonic mesoderm development. Importantly, TSP-14A and TSP-14B exhibit distinct subcellular localization patterns. TSP-14A is localized apically and on early and late endosomes. TSP-14B is localized to the basolateral cell membrane. We further identified a di-leucine motif within the N-terminal 24 amino acids of TSP-14B that serves as a basolateral membrane targeting sequence, and showed that the basolateral membrane localization of TSP-14B is important for its function. Our work highlights the diverse and intricate functions of TspanC8 tetraspanins in C. elegans, and demonstrates the importance of dissecting the functions of these important proteins in an intact living organism. Author summaryTetraspanin proteins are a unique family of highly conserved four-pass transmembrane proteins in higher eukaryotes. Abnormal expression of certain tetraspanins is associated with various types of diseases, including cancer. Understanding the functions of different tetraspanin proteins in vivo is crucial in deciphering the link between tetraspanins and their associated disease states. We have previously identified two tetraspanins, TSP-12 and TSP-14, that share redundant functions in regulating multiple aspects of C. elegans development. Here we show that TSP-14 has two protein isoforms. Using CRISPR knock-in and knock-out technology, we have found that the two isoforms share unique, as well as overlapping expression patterns and functions. Furthermore, they exhibit distinct subcellular localization patterns. Our work highlights the diverse and intricate functions of tetraspanin proteins in a living multicellular organism, and demonstrates that protein isoforms are another mechanism C. elegans uses to increase the diversity and versatility of its proteome.