A lipid transfer protein knockout library reveals ORP9-ORP11 dimer mediating PS/PI(4)P exchange at the ER-trans Golgi contact site to promote sphingomyelin synthesis

Reviewer #1 (Public Review):

Summary:
In this well-designed study, the authors of the manuscript have analyzed the impact of individually silencing 90 lipid transfer proteins on the overall lipid composition of a specific cell type. They confirmed some of the evidence obtained by their own and other research groups in the past, and additionally, they identified an unreported role for ORP9-ORP11 in sphingomyelin production at the trans-Golgi. As they delved into the nature of this effect, the authors discovered that ORP9 and ORP11 form a dimer through a helical region positioned between their PH and ORD domains.

Strengths:
This well-designed study presents compelling new evidence regarding the role of lipid transfer proteins in controlling lipid metabolism. The discovery of ORP9 and ORP11's involvement in sphingolipid metabolism invites further investigation into the impact of the membrane environment on sphingomyelin synthase activity.

Weaknesses:
There are a couple of weaknesses evident in this manuscript. Firstly, there's a lack of mechanistic understanding regarding the regulatory role of ORP9-11 in sphingomyelin synthase activity. Secondly, the broader role of hetero-dimerization of LTPs at ER-Golgi membrane contact sites is not thoroughly addressed. The emerging theme of LTP dimerization through coiled domains has been reported for proteins such as CERT, OSBP, ORP9, and ORP10. However, the specific ways in which these LTPs hetero and/or homo-dimerize and how this impacts lipid fluxes at ER-Golgi membrane contact sites remain to be fully understood.

Regardless of the unresolved points mentioned above, this manuscript presents a valuable conceptual advancement in the study of the impact of lipid transfer on overall lipid metabolism. Moreover, it encourages further exploration of the interplay among LTP actions across various cellular organelles.

Reviewer #2 (Public Review):

Summary:
The authors set out to determine which lipid transfer proteins impact the lipids of Golgi apparatus, and they identified a reasonable number of "hits" where the lack of one lipid transfer protein affected a particular Golgi lipid or class of lipids. They then carried out something close to a "proof of concept" for one lipid (sphingomyelin) and two closely related lipid transfer proteins (ORP9/ORP11). They looked into that example in great detail and found a previously unknown relationship between the level of phosphatidylserine in the Golgi (presumably trans-Golgi, trans-Golgi Network) and the function of the sphingomyelin synthase enzyme. This was all convincingly done - results support their conclusions - showing that the authors achieved their aims.

Impact:
There are likely to be 2 types of impact:
(I) cell biology: sphoingomyelin synthase, ORP9/11 will be studied in the future in more informed ways to understand (a) the role of different Golgi lipids - this work opens that out and produces more questions than answers (b) the role of different ORPs: what distinguishes ORP11 from its paralogy ORP10?
(ii) molecular biochemistry: combining knockdown miniscreen with organelle lipidomics must be time-consuming, but here it is shown to be quite a powerful way to discover new aspects of lipid-based regulation of protein function. This will be useful to others as an example, and if this kind of workflow could be automated, then the possible power of the method could be widely applied.

Strengths:
Nicely controlled data;
Wide-ranging lipidomics dataset with repeats and SDs - all data easily viewed.
Simple take-home message that PS traffic to the TGN by ORP9/11 is required for some aspect of SMS1 function.

Weaknesses:
Model and Discussion:
No idea about the aspect of SMS1 function that is being affected. Even if no further experiments were carried out, the authors could discuss possibilities. One might speculate what the PS is being used for. For example, is it a co-factor for integral membrane proteins, such as flippases? Is it a co-factor for peripheral membrane proteins, such as yet more LTPs? The model could include the work of Peretti et al (2008), which linked Nir2 activity exchanging PI:PA (Yadav et al, 2015) to the eventual function of CERT. Could the PS have a role in removing/reducing DAG produced by CERT?

Reviewer #3 (Public Review):

Summary:
The authors developed a lipid transfer protein knock-out library to identify lipid transfer proteins with roles in lipid homeostasis/metabolism. They investigated one of their hits, the ORP9/ORP11 dimer, which they found affects sphingomyelin synthesis. Further. they found that ORP9/11 localizes to ER-Golgi contact sites via interactions between a known FFAT motif in ORP9, which can interact with the ER protein VAP, and the PH domains of ORP9 and ORP11 that target PI4P at the Golgi. They showed defects in Golgi PI4P and PS levels when ORP9 or 11 were dysfunctional, supporting but not demonstrating that ORP9/11 might exchange PI4P and PS at these contact sites. Their in vitro data indicates that both ORP9 and 11 can transfer PS. They do not assess whether either protein can transfer PI4P (although there is a very nice recent paper by He et al et You, PMID 36853333, showing that ORP9 can transfer PI4P in vitro), and they do not assess the consequences of heterodimerization on either PS or PI4P transfer. The mechanisms by which PI4P/PS level perturbations affect sphingomyelin synthesis remain unclear.

Strengths:
The authors have developed an LTP knock-out library that might generate hypotheses regarding lipid metabolism, although defects are not unexpected and mechanisms will be difficult to work out--as, in fact, evidenced by this manuscript.

The OPR9/11 localization data and imaging studies are well done; this is the first more comprehensive characterization of the ORP9/11 heterodimer, which was discovered in 2010.

Weaknesses:
A major flaw is that the authors claim to but do not, in fact, provide evidence of PS/PI4P counter exchange in vitro. That the presence of PI4P on the acceptor liposomes accelerates PS transfer in the in vitro assays is not proof that there is a counter exchange. In fact, since the rate-determining step in the transfer reactions is lipid transfer between membrane and transfer protein and this depends on the association of the transfer protein with donor and/or acceptor liposomes, a more likely explanation for the more efficient transfer in the presence of PI4P is that PI4P allows for longer association of lipid transfer protein with acceptor liposomes. To show the plausibility of the counter-exchange idea as applied to the ORP9/ORP11 heterodimer, the authors would need to show that it can transfer PI4P. (The work by He et al et You, 2023, mentioned above, is a very nice study that the authors might use as a model.)

Mechanistic insights from the study are limited. How does a PI4P/PS imbalance affect sphingomyelin synthesis?

The ORP9/11 heterodimer seems to behave very much like ORP9/ORP10 heterodimer, including in its localization and dimerization mode. Is ORP9/11 just another version of 9/10? I wonder whether discussions of whether they are redundant or what their different roles are might be in order. There is little mechanistic or conceptual novelty arising from this study.

A minor point, but the statement (p2., lines 19-20) that "vesicular trafficking contributes only to a small portion of lipid trafficking" is not correct and raises eyebrows. What is more correct is that protein-mediated lipid transfer ALSO plays an important role in lipid transfer. It might even be said that LTP-mediated lipid transfer is critical in fine-tuning membrane lipid composition, including of phosphoinositides.