Phosphoinositides (PI) is an essential signaling molecule as it is a component of a diversified number of cellular functions, such as growth, differentiation, migration, and endocytosis. In this paper, Stefan and his group hypothesized that monophosphorylated PI4P at the PM is linked to Sac1 at the ER with the help from PI4P binding proteins. Their hypothesis is based on the assumption that ER Sac1 controls PM PI4P at the PM/ER membrane contact sites. Previous research reported ∆Sac1 leads to accumulation of PI4P at PM. Sac1 was shown to regulate PI4P at ER and Golgi, however the linkage between the two at the PM was yet investigated. Therefore, this paper is significant by identifying oxysterol-binding homology (Osh) proteins as the missing link connecting Sac1 PI phosphatase activity to PI4P at PM.
To approach the hypothesis, this group utilized three key major experimental procedures: PI analysis, protein-binding assays and phosphatase assays. First, to measure the level of PI4P, PI analysis was conducted. Yeast was harvested and its PI was labeled with myo-[2-3H]-inositol, followed by HPLC separation and radio-detection to quantify PI4P. Next, protein-binding assays were conducted to determine interaction. Proteins with GST-tag was immobilized on sepharose 4B and incubated with potential interactors. The bead was then washed and bound protein was eluted for immunoblotting to identify potential interaction. Crosslinking was also used to isolate indirect complex. Furthermore, phosphatase assay was conducted to measure the phosphate released from the mixture of microsomes and liposomes/PI4P, or proteins and liposomes/PI4P, depending whether the assay is for microsome phosphatase or Sac1 phosphatase. For Sac1 assay, purified proteins was added to liposome/PI4P, then the release of phosphate was measured by addition of Biomoi Green, followed by quantification by a photometer.
This group started the investigation by characterizing the domains of Osh proteins. Osh 1,2,3 are similar to mammalian OSBP which contains a PH domain that bind to PI4P. Osh3 is localized to PM/ER contact sites in response to the level of PI4P at PM. The FFAT motif of Osh3 can rescue the growth defect of Osh∆:CenOshTS. In addition, Osh3 interacts with VAP proteins, Scs2 and Scs22, via FFAT motif. The group continued to look at the connection between Osh proteins and PI4P level. PI4P level was 6-7 fold higher in Osh∆:CenOsh4 and Osh∆:CenOsh4TS from baseline, which indicates that Osh proteins have a regulatory role in PI4P level. This is also consistent with the elevated PI4P level from Osh2∆ Osh3∆ double mutant. Osh∆:CenOsh4TS showed 18 fold increase in PI4P level, suggesting that Osh proteins also regulate the activity of Sac-1. Furthermore, in ∆Scs2, ∆Scs22 double mutant, PI4P level was up by 2.9 fold and PI4P was stabilized at the PM. Microsomes without these VAP proteins showed reduced Sac1 activity and PI4P turnover, which can be rescued by Osh3. This indicated that VAP proteins regulate PM PI4P and Sac1 activity. OSBP-related sterol-binding domain (ORD) of Osh3 and Osh4 was able to activate Sac1. Osh43E (R236E, R242E, R243E) mutant showed impaired ability to bind to PI and stimulate Sac1 activity. Comparing to baseline, PI4P level was 17 fold higher in Osh∆:CenOsh4TS strain coexpressing Osh3E; and ORD mutants of Osh4 also showed 19-21 fold increase in PI4P, indicating that PI and sterol binding are essential for Osh4-mediated PI4P metabolism. Osh7 and Osh3 were found to interact with Sac1 through transient interaction or indirect interaction. Interaction between some Osh proteins and Sac1 is dependent on the interaction between ORD and Sac1.
Stefan and his group had presented a convincing story. I agree that Osh proteins regulate phosphoinositide metabolism at the contact sites between ER and PM. For the future direction, we have to (i) link this finding to a bigger picture, as in how the regulation at PM influences the downstream pathways, and (ii) distinguish PM PI4P from PI4P localized elsewhere. In order to do so, overexpress Osh proteins in cells, followed by immunoblotting for markers of downstream pathways. By establishing downstream expression profiles, we would be able to know what the key pathways are regulated by PM PI4P, but not other PI4P that are localized somewhere else.