Utilizing a nanobody recruitment approach for assessing serine palmitoyltransferase activity in ER sub-compartments of yeast

  1. Bianca M. Esch
  2. Stefan Walter
  3. Oliver Schmidt
  4. Florian Fröhlich

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Abstract

Sphingolipids (SP) are one of the three major lipid classes in eukaryotic cells and serve as structural components of the plasma membrane. The rate-limiting step in SP biosynthesis is catalyzed by serine palmitoyltransferase (SPT). In yeast, SPT consists of two catalytic subunits (Lcb1 and Lcb2), a regulatory subunit (Tsc3), negative regulators (Orm1 and Orm2), and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1, collectively known as the SPOTS complex. Regulating SPT activity enables cells to adapt SP metabolism to changing environmental conditions. Therefore, the Orm proteins are phosphorylated by two signaling pathways originating from either the plasma membrane localized target of rapamycin (TOR) complex 2 or the lysosomal/vacuolar TOR complex 1. Moreover, uptake of exogenous serine is necessary for the regulation of SP biosynthesis, which suggests the existence of differentially regulated SPT pools based on their intracellular localization. However, tools for measuring lipid metabolic enzyme activity in different cellular compartments are currently not available. We have developed a nanobody recruitment system that enables the re-localization of the SPOTS complex to the nuclear or peripheral ER. By combining this system with sphingolipid flux analysis, we have identified two distinct active SPT pools in cells. Our method thus serves as a new and versatile tool to measure lipid metabolism with sub-cellular resolution.

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  1. Review Commons

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    Reply to the reviewers

    The authors do not wish to provide a response at this time.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors of this study utilize a novel nanobody-based technique to specify the location of the SPT complex to either the peripheral or nuclear membrane-associated endoplasmic reticulum membranes. Considering the potential importance of sub-ER compartmentalization on metabolic enzymes of the ER, this is a novel and useful approach. The studies are, with the minor exceptions noted below, comprehensive and very well executed and documented. The authors have combined genetic, proteomic, lipidomic, and flux experimental approaches to test whether sub-ER compartmentalization affects the function and regulation of the SPT complex. The results are, for the most part, negative, although there does seem to be some effect on the overall activity of the SPT complex as measured with flux analysis. Overall, while the authors do not detect dramatic effects on SPT complex localization, the technical advance using tethered nanobodies to direct complex localization, and the complementary approaches to testing SPT function and regulation, will be useful to workers in the sphingolipid field.

    Minor points:

    The results with YPK1-linker-CAAX are confusing. This construct does not result in Orm2 phosphorylation with heat shock, whereas endogenous YPK1 does. Yet it can support viability even without Orm deletion. In other words, this tethered construct appears functional in viability assays, but not in a biochemical assay.This discrepancy is not discussed by the authors. The manuscript would be improved by a discussion by the authors that addresses this issue. It is not clear why the figure legend to Figure 2 suggests that Ypk1 regulates Orms mainly in the peripheral ER. Considering that WT Ypk1 is more efficient than CAAX tethered YPK1, this statement does not seem supported. Perhaps the authors can elaborate on how they came to this conclusion.

    The figures depicting Orm phosphorylation (Figure 1e, f Figure 2d,e, Figure 6 b,c) should be improved. The resolution of two forms is not sufficient in Figure1 and 2. The use of Phos-Tag might solve this issue. It would be helpful to the reader to include arrows that indicate the phosphorylated and unphosphorylated forms of Orm. Quantitation of these gels is essential.

    Lines 318 and 319. Figure 6e and 6f are referred to. The correct assignment is 6f and 6g.

    Referees cross-commenting

    I agree with Reviewer #2's assessment that some of the conclusions are over stated. While Reviewer #2 is correct that the advances in this manuscript are modest, this is principally because expected differences in the function and regulation of the SPT in different ER sub-domains did not materialize. This may be disappointing, but is still important to document

    Significance

    This is a very well performed study, utilizing a variety of approaches to test whether localization of the SPT complex impacts on it activity and regulation. With very minor exceptions, it is well executed and documented.

    The advances reported here are two-fold. First, the authors introduce a novel approach using nanobodies that are tethered to distinct regions of the yeast endoplasmic reticulum to localize intact and unmodified complexes to distinct locations. This could be a very useful tool in other contexts to examine the role of subcellular compartmentalization in the function of enzymes and signaling components. This targeting system is well characterized in this study. The second advance, utilizing this targeting system, is that localization of the SPT complex to distinct subcompartments of the ER has minimal effects on regulation, and observable, but relatively minor effects on SPT function in terms of sphingolipid production. While a positive result would have been more exciting, negative results can be equally informative.

    This study will be of interest to workers in the signaling and metabolic fields that may utilize this unique targeting strategy. It will also be of interest to the sphingolipid community.

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    Referee #2

    Evidence, reproducibility and clarity

    This manuscript uses a combination of immunoblotting, microscopy, and MS-based lipidomics to study sphingolipid synthesis in S. cerevisiae. The authors recently published a paper demonstrating that exogenous serine taken from the medium is preferentially used to generate LCBs. Given multiple levels of regulation of the SPT complex, the authors postulate that SPT in different sub-compartments of the ER could be differently regulated or at least have variable activities. Using a nanobody capture approach, they can restrict SPT activity to the peripheral ER and nuclear ER. Using this model, they investigate the role of Orm phosphorylation and SPT activity in response to 5-min heat shock. Phosphorylation does not seem to be a key element of the regulation. Ultimately, this is a collection of experiments without a clear story. In the end, the only take-home message I can find is that peripheral SPT is able to use exogenous heavy Serine as a substrate better than nuclear SPT.

    Page 4. "We had previously demonstrated that increased de novo LCB biosynthesis is directly dependent on the uptake of exogenous serine through the general amino acid permease Gnp1. Consistent with our previous findings, deletion of GNP1 resulted in a blunted heat shock response, while deletion of the endogenous serine biosynthesis pathway (ser2) had no effect on LCB biosynthesis". This claim is too strong. If they feel this strongly, the authors should test a gnp1 agp1 double mutant. The alternative explanation is that to support the rapid increase in SPT activity, the Lcb1-Lcb2 enzyme use both a pre-existing cytoplasmic pool of serine and exogenous serine.

    If cells are grown for 1 generation with heavy serine to label the cytoplasmic pool of serine and then cells as shifted to serine-free media and heat shock is induced is there any difference in LCB synthesis between allSPT, nSPT and pSPT?

    It would be worth re-doing some of these experiments in rtn1 rtn2 yop1 yeast triple mutant (Stefan...Emr Cell 2011) or the delta super-tether mutant (Quon...Menon PLOS Biology 2016) both of which have substantially less cortical ER.

    The authors make strong claims that are not supported by the data. Further, the presentation of the data is not optimal, and much of the data is qualitative, not quantitative. Too much of the data is presented as fold-change and i believe that the base-line LCB levels may change. The raw data should be included in a supplement excel file.

    The blots of FLAG-tagged Orm1 and Orm2 are a critical part of this manuscript, but the data is not compelling in most figures. There is a lack of quantitation and replication. On the surface I agree with the authors that the phosphorylation is hard to align with the stimulation, but a more rigorous analysis is needed. Additionally, does the expression of an Orm2 mutant without the Ypk1 phosphorylation sites prevent the heat shock-induced increase in LCBs?

    If this remains in the manuscript, the difference between ypk2 Ypk1-CaaX and ypk2 Ypk1-103aa-CaaX should be better highlighted in the main document. However, this whole course of experiments is problematic, and the authors' narrative changes to accommodate the findings; the arguments aren't internally consistent. Ypk is essential to regulate Orms. Ypk1-linker-CaaX can regulate Orms, Ypk1-linker can't increase Orm2 phosphorylation. Furthermore, a single prenyl group is not sufficient to restrict the localization of a protein. If there are other targeting motifs in addition to the last 4 amino acids this should be indicated. Ultimately, this is all negative data. Without a better interrogation of Orm phosphorylation it seems to have little value.

    The "Orm proteins mediate SPT upregulation after heat shock" should be re-worded. The orm1 orm2 mutant already has a 25-fold increase in LCBs, and heat shock is unable to stimulate SPT activity any further. It suggests that heat shock is directly inhibiting Orm proteins which in turn removes the feedback inhibition on the SPT.

    Figure 7b - the Y-axis scale needs to be corrected. A break should be inserted to alert the reviews of the scale's narrow range.

    Is GFP-Orm1 and Orm2 functional? Both are more abundant in the nER than the peripheral.

    Most papers I have read use "SL" as the abbreviation for sphingolipids, not SP. Consistency would be nice for readers.

    Significance

    The work appears technically sound but there are issues with the presentation of the data. Lack of raw data for the lipidomics, lack of quantitation.

    The findings here are rather modest and much of the data is inconclusive or suggests certain pathways aren't involved rather than defining a clear mechanism.

    This is an incremental finding that supports the 2020 PLOS genetics paper from the same authors. The target audience for this would be people interested in sphingolipid metabolism in yeast.

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    Referee #1

    Evidence, reproducibility and clarity

    This is a nice paper showing that the intracellular location of the SPOTS complex affects SL de-novos synthesis. The experiments are well designed including a comprehensive set of controls. I just have a few minor points: It is hard for me to see the differences in ORM phosphorylation on the blots. Does phosphorylation refer to the more intense upper band (Fig 1e+f)? Given the general variability of WBs it might be better to demonstrate the differences in ORM phosphorylation with a more specific and quantitative method? e.g by a phosphoprotein stain or (better) by targeted (phospho)proteomics.

    Significance

    Unfortunately, the discussion focusses mostly on technical aspects of the method. However, the biological observations concerning the regulation of the SL metabolism itself are interesting and relevant too. They should be discussed in more detail, which might also be of interest to a more general readership. Concerning the factors involved in regulating SL de-novo synthesis, the authors did not mention CERT which also contributes to the regulation of SL de-novo formation (PMID: 36976648) and connects to SacI which is a component of the SPOTS complex.
    Why does yeast have two ORM isoforms and to which extend are they redundant? The authors see functional difference between the two ORM isoforms, which could be discussed in more depth. It might also be interesting to interlink the findings to the mammalian system, which is based on three ORMDL isoforms, and appear not to be regulated by phosphorylation. Another aspect that could be discussed in this contet is the observation that mammalian SPT appears preferentially located at MEM contact sites, which indicates a special role of SL de-novo synthesis at this location (PMID: 34785538). However, overall this is a well done paper.

  5. Version published to 10.1101/2023.03.29.534722v1 on bioRxiv