Investigating Env9's Mechanism of Membrane Insertion in Saccharomyces cerevisiae

Abstract

Tail‐anchored proteins have vital roles within the cell such as vesicular transport and apoptosis, and often times their specific membrane localization is crucial to their function. These proteins have hydrophobic regions near their C‐terminus, making them identifiable to protein insertion machinery. One pathway which targets C‐tail anchored proteins is the well‐characterized Guided Entry of Tail‐Anchored Protein (GET) complex, consisting of Get1‐5 and Sgt2. Interestingly, a second mechanism has been proposed named the SRP‐independent (SND) pathway which similarly targets tail‐anchored proteins. Our protein of interest, Env9, is one of four novel endosome to vacuole interface (ENV) gene products and our data suggests it possesses a hydrophobic region near its C‐terminus. Env9 localizes to lipid droplet membranes however, its insertion mechanism is unknown. We hypothesize Env9 is targeted to membranes by either GET or SND pathways because of both its c‐terminal hydrophobic domain and its interactions with components of both complexes. Negative genetic interactions with both ENV8 (GET4) and with ENV10 (SND2) have been reported in systematic studies and have been observed in our laboratory. In order to investigate these interactions, we used both microscopic and biochemical approaches. Env9 was tagged with either GFP (green fluorescent protein) under an inducible galactose promoter or HA (hemagglutinin) under a constitutive PGK promoter to analyze localization in GET and SND mutants via confocal microscopy and western blot analysis, respectively. Preliminary microscopy results show slight disruption in GET mutants whereas SND mutants appear to have wild‐type Env9 localization patterns. In preliminary western blot analysis, tagged double mutants Δget4/env9 and Δsnd2/env9 continue to localize Env9‐HA to the P13 pellet that includes lipid droplets, but also vacuoles and aggregated protein complexes. These results suggest Env9 may be targeted to membranes via the GET complex and specifically implicate the GET complex in the process of insertion of Env9 into the ER membrane. These moderate phenotypes may also suggest compensation by one complex in the absence of the other in order to accurately target vital C‐tail anchored proteins. Additionally, the compensatory effect may be further stimulated by the highly overexpressed constructs used in these studies. We are currently exploring the insertion of Env9 expressed at native levels. Additionally, we are examining Env9 localization in GET double and triple mutants, as well as determining protein‐protein interactions via other biochemical approaches such as co‐immunoprecipitation.Support or Funding InformationAcknowledgements: This project was supported by NIH AREA #R‐15 GM85794‐02, NSF‐MRI grant #DBI0722757 for confocal microscopy, and NIH 2R25GM071638‐09A1 for the Research Initiative for Scientific Advancement Fellowship funding. We'd like to thank Dr. Maya Schuldiner at the Weizmann Institute of Science for her donations of the GET and SND mutants.