Endoplasmic Reticulum Tubule Protein Reticulon 4 Associates with the Legionella pneumophila Vacuole and with Translocated Substrate Ceg9

Intracellular growth of Legionella pneumophila occurs in a replication vacuole constructed by host proteins that regulate vesicular traffic from the host endoplasmic reticulum (ER). This process is promoted by a combination of approximately 300 Icm/Dot translocated substrates (IDTS). One of these proteins, Ceg9, was previously identified in a screen for L. pneumophila IDTS that manipulate secretory traffic when overexpressed in yeast. Using ectopic expression of Ceg9 in mammalian cells, we demonstrate that Ceg9 interacts with isoforms of host reticulon 4 (Rtn4), a protein that regulates ER tubule formation. Binding occurs under conditions that prevent association with other known reticulon binding proteins, arguing that Ceg9 binding is stable. A tripartite complex was demonstrated among Rtn4, Ceg9, and atlastin 1, a previously characterized reticulon interacting partner. The binding of Ceg9 to Rtn4 was not due to bridging by atlastin 1 but resulted from the two interacting partners binding independently to reticulon. When Ceg9 is ectopically expressed in mammalian cells, it shows a localization pattern that is indistinguishable from that of Rtn4, perhaps due to interactions between and similar structural features of the two proteins. Consistent with Rtn4 playing a role in the formation of the Legionella-containing vacuole, it was recruited to almost 50% of the vacuoles within 20 min postinfection. Our studies suggest that L. pneumophila proteins interact with ER tubules at an early stage of replication vacuole formation.