Supplementary Material for: The Nonaspanins TM9SF2 and TM9SF4 Regulate the Plasma Membrane Localization and Signalling Activity of the Peptidoglycan Recognition Protein PGRP-LC in Drosophila

Transmembrane 9 (TM9) proteins, or nonaspanins, are a family of proteins conserved throughout evolution and characterized by 9 transmembrane domains. In <i>Drosophila</i>, TM9 superfamily protein member 4 (TM9SF4) and its closest paralogue, TM9SF2, contribute to phagocytosis of various types of particles, while TM9SF4 displays non-redundant requirement in Gram-negative bacteria engulfment. In addition, the two TM9 proteins control the actin cytoskeleton in larval haemocytes and in <i>Drosophila </i>S2 cells. Here, we show that TM9SF4 and TM9SF2 co-immunoprecipitate with the peptidoglycan recognition protein (PGRP)-LC, which triggers the <i>Drosophila</i> immune response to bacterial infection. Furthermore, both TM9 proteins co-localize with this receptor in intracellular vesicles and at the plasma membrane in <i>Drosophila</i> S2 cells in culture and in the fly fat body. Silencing <i>TM9SF4 </i>prevents plasma membrane localization of PGRP-LC, whereas silencing <i>TM9SF2 </i>does not, which may account for the non-redundant role of TM9SF4 in phagocytosis of Gram-negative bacteria. Finally, we provide a set of data suggesting that TM9 proteins can prevent inappropriate signalling from the unstimulated receptor.