N-terminal chimaeras with signal sequences enhance the functional expression and alter the subcellular localization of heterologous membrane-bound inorganic pyrophosphatases in yeast
Authors: Rocío Drake
José R Pérez-Castiñeira
Aurelio Serrano
Journal: Biochemical Journal (2010)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Saccharomyces cerevisiae; membrane proteins; heterologous expression; signal sequences; proton-translocating inorganic pyrophosphatase
Affiliations: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Centro de Investigaciones Cientificas Isla de la Cartuja, Avda. Americo Vespucio, 49, E-41092 Sevilla, Spain., Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Centro de Investigaciones Cientificas Isla de la Cartuja, Avda. Americo Vespucio, 49, E-41092 Sevilla, Spain

Abstract:  Expression of heterologous multispanning membrane proteins in Saccharomyces cerevisiae is a difficult task. Quite often, the use of multicopy plasmids where the foreign gene is under the control of a strong promoter does not guarantee efficient production of the corresponding protein. In the present study, we show that the expression level and/or subcellular localization in S. cerevisiae of a heterologous type of multispanning membrane protein, the proton-translocating inorganic pyrophosphatase (H+-PPase), can be changed by fusing it with various suitable N-terminal signal sequences. Chimaeric proteins were constructed by adding the putative N-terminal extra domain of Trypanosoma cruzi H+-PPase or the bona fide signal sequence of S. cerevisiae invertase Suc2p to H+-PPase polypeptides of different organisms (from bacteria to plants) and expressed in a yeast conditional mutant deficient in its cytosolic PPi hydrolysis activity when grown on glucose. Chimaeric constructs not only substantially enhanced H+-PPase expression levels in transformed mutant cells, but also allowed functional complementation in those cases in which native H+-PPase failed to accomplish it. Activity assays and Western blot analyses demonstrated further the occurrence of most H+-PPase in internal membrane fractions of these cells. The addition of N-terminal signal sequences to the vacuolar H+-PPase AVP1 from the plant Arabidopsis thaliana, a protein efficiently expressed in yeast in its natural form, alters the subcellular distribution of the chimaeras, suggesting further progression along the secretory sorting pathways, as shown by density gradient ultracentrifugation and in vivo fluorescence microscopy of the corresponding GFP (green fluorescent protein)-H+-PPase fusion proteins. 

Retracted: No

PubPeer comments:
 Figure 1C and 2A: much more similar than expected (images marked with boxes of different colors). Moreover, cells and growing conditions are different. Could the authors please comment? ![file] , 
 Figure 4A: images marked with pink boxes seem to be the same image (after vertical flip). ![file] , 
 Figure S1A: images marked with green boxes seem to be the same image (after vertical flip). ![file] , 
 The text written below has been read by all authors and they all agree with its content **Figure 1C and Figure 2A central panel** Cells are not that different: W303 is the parental strain of YPC3, the only change between both of them is the insertion of the GAL1 promoter controlling the expression of IPP1 in the latter. In my view, there is no surprise in these pictures because, although W303 (and most yeast strains) grows faster in glucose than in galactose we decided to wait as long as necessary in order to make sure that YPC3 had no growth whatsoever in glucose. The alternative was that YPC3 might have grown slower that W303 in the presence of this carbon source; however, we were looking for a u201call or nothingu201d response and wanted to be certain that this was indeed the case. Moreover, we had previously generated a mutant with the same phenotype as YPC3, albeit using a different experimental approach (Perez-Castiu00f1eira et al., 2002). The former had the tendency to produce revertants after a few days, therefore, it was important for us to check that the insertion introduced in YPC3 was perfectly stable. For this reason, we incubated the plates at 30u00baC until all cells reached the equivalent to a late stationary phase in both carbon sources. If we had really wanted to see a different growth rate between W303 and or YPC3 transformed with IPP1 grown in galactose with respect to glucose, we should had made a growth curve; however, this was not considered necessary by the editor or the referees, after all, the point here was the recovery of growth in glucose (not in galactose) when our mutant was transformed with plasmids bearing membrane-bound PPases. **Figure 2A left panel** It seems that we made a mistake when we elaborated this figure for the paper. The results shown in this manuscript were part a a Ph.D. thesis that it was going to be submitted in 2010 (although the Ph. D student finally quit, moved to the private industry, and never got her Ph. D.). A pdf with a thesis chapter (generated on April 2nd, 2009) in which you can see the original image (Figure 5 of this chapter) is available at: https: www.dropbox.com s 7zv7wg22cjzdusk Complementacion%20H%2BPPasas%20en%20YPC3.pdf?dl=0 You can see that the result was identical to that shown in Figure 2A. **Figures 4A and S1A** We were so interested in the growth on glucose (the proof of the functional complementation of yeast soluble PPase by different chimaeric membrane PPases) that we might have made some mistakes with the drops on galactose, where the growth was always identical. We did these experiments so many times with so many different transformants that, at the end, we had tens of plates with tens of spots each one. Galactose plates were particularly u201cboringu201d because we never saw any significant difference in growth on this carbon source with any of the constructions tested (you can see that in the pdf I attach -Figures 5, 8, 9, 10, 11 and 14-). There were three people involved in taking photos and we had images with plates upside up and upside down, therefore, sometimes we had to flip the images and it may have been the case that we made mistakes when we did it. In any case, this was absolutely unintended and does not change the results whatsoever: we have been using YPC3 (and similar mutants like YPC5 and YPC6 -see references-) for nearly 20 years and all of them grow exactly the same as the parental strain W303 in galactose. The reason for this is clear to us after so much time working with PPases in yeast: the level of cytosolic PPase activity in the cytosol in wild type strain (W303) grown with any carbon source, and in YPC3-like mutants in galactose is way above cellular needs, so that the rate of production of PPi is the actual bottleneck of PPi hydrolysis and not the level of PPase. The point of our mutants is that we were able to bring IPP1 expression down to negligible levels simply by changing the carbon source. The consequence was that cells stopped growing, thereby demonstrating the essentiality of IPP1. This was appreciated by many other scientific groups that rapidly asked us to provide YPC3 for their own work. We did make it available subjected to certain conditions and with the correspondent MTA agreement, as YPC3 is under patent. None of these group have ever complained about YPC3 phenotype, actually, two of them have published papers that confirm our results with this mutant (see below). **References** Perez-Castineira JR, Lopez-Marques RL, Villalba JM, Losada M, Serrano A. Functional complementation of yeast cytosolic pyrophosphatase by bacterial and plant H+-translocating pyrophosphatases. Proc Natl Acad Sci U S A. 2002;99(25):15914-15919. doi:10.1073 pnas.242625399 Pu00e9rez-Castiu00f1eira JR, Hernu00e1ndez A, Drake R, Serrano A. A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast. Biochem J. 2011 Jul 15;437(2):269-78. doi: 10.1042 BJ20110447. PMID: 21612578. Hernu00e1ndez A, Serrano-Bueno G, Perez-Castiu00f1eira JR, Serrano A. 8-Dehydrosterols induce membrane traffic and autophagy defects through V-ATPase dysfunction in Saccharomyces cerevisae, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2015, 1853, Issue 11, Part A, 2945-2956, ISSN 0167-4889, https: doi.org 10.1016 j.bbamcr.2015.09.001. Pu00e9rez-Castiu00f1eira JR, Serrano A.The H+-Translocating Inorganic Pyrophosphatase From Arabidopsis thaliana Is More Sensitive to Sodium Than Its Na+-Translocating Counterpart From Methanosarcina mazei. Frontiers in Plant Science 2020, 11, 1240 https: www.frontiersin.org article 10.3389 fpls.2020.01240. DOI=10.3389 fpls.2020.01240 t Papers from groups that have used YPC3 mutant and have reported that the mutant has the appropriate phenotype: Emily M. Coonrod, Laurie A. Graham, Lindsay N. Carpp, Tom M. Carr, Laura Stirrat, Katherine Bowers, Nia J. Bryant, Tom H. Stevens. Homotypic Vacuole Fusion in Yeast Requires Organelle Acidification and Not the V-ATPase Membrane Domain. Developmental Cell, 2013, 27(4), 462-468. ISSN 1534-5807. https: doi.org 10.1016 j.devcel.2013.10.014. Desfougu00e8res, Y., Vavassori, S., Rompf, M. et al. Organelle acidification negatively regulates vacuole membrane fusion in vivo. Sci Rep 6, 29045 (2016). https: doi.org 10.1038 srep29045 , 
 #4 Dear Dr. Pu00e9rez-Castiu00f1eira, thank you for your reply and detailed explaination. Since you honestly recognized these are all mistakes, may I ask you whether you are planning to notify the journal in order to issue a correction? Best regards. , 
 #4 One more thing. Given the number and quality of these mistakes, I believe a correction would be mandatory. Moreover, I find quite remarkable that, with  tens of plates available, three people taking photos were so unlucky in making so many unintended mistakes, and later noone spotted them. , 
 I was not thinking of notifying the journal because, as I told you in my previous message, these mistakes do not mean anything in practical terms: the work performed by our group along two decades, as well as that done by other groups independently demonstrates that YPC3 and all the mutants generated with the same strategy (YPC5 and YPC6) grow equally well in the presence of galactose and all of them stop growing in the presence of glucose. Upon transformation with plasmids bearing foreign PPases (either soluble or membrane-embedded) they keep on growing equally well on galactose (positive control) but the recovery of growth in glucose depends on the particular foreign PPase that it s been expressed (see figures attached). This is the whole point of our system and the reason why so many groups have used it. With respect to your final remarks, I have to say that we are talking about experiments that took place more than a decade ago because, although the paper was published in 2010, the work included there had been going on for more than five years before publication. Along those years, I guess we took pictures of more than a hundred plates, each of them having an average of 25-30 spots, because we checked the ability of tens of soluble and membrane-bound PPases from different sources to functionally complement the yeast soluble PPases and, of course, we had to make sure that the results were reproducible, therefore, the experiments had to be repeated at least three times. Only a few of these pictures were selected for this particular paper, which means a lot of work for three people and, in my view, increases the probability of mistakes. In any case, I can only guess what might have happened but I cannot be certain about what actually happened. I also want to add that, although we are a small group working on a relatively unknown scientific field, we have our reputation and have published in top journals for more than twenty years without having any problems or complaints. Sometimes I have spotted small mistakes after papers, Ph. D. theses, and even textbooks (*) have been published, even though the manuscripts were presumably read by authors, supervisors, editors and or reviewers. This does not mean that those mistakes were intended. (*) I have sent emails to the authors and editors of Lehningeru2019s Principles of Biochemistry to tell them that they had made a mistake. This was gratefully acknowledged in the following edition of the book (5th edition, page xiv of Preface, ISBN-13: 978-0-7167-7108-1). ![file] ![file] ![file] ![file] , 
 #7 Dear Dr. Pu00e9rez-Castiu00f1eira, thank you for your second reply. * these mistakes do not mean anything in practical terms *: sorry but I do strongly disagree with this line of argumentation. **These five mistakes do mean that five images are describing something that is actually something else. Five significant mistakes are affecting 3 out of 6 main figures and one supplementary figure.** You admitted these are mistakes, so why not issuing a correction? If I got it right, you posted images of other plates, so It would be an easy job. I wish to believe you have a reputation, and a correction would preserve it rathar than undermine it. Literature should be corrected as soon as mistakes are proven to be so, and highest stardard of rigour and accuracy should be pursued even after first publication. , 
 #4 Iu2019m not sure if this issue is pertinent to this paper, however Figure 5 from the thesis available at the Dropbox link provided above has some repeated bands, described differently. *** False color ![file] *** Original ![file] *** ![file] , 
 Fair enough, we don t agree I sent a DRAFT of a Ph. D. thesis that it was not defended, because the Ph. D. student quit to the private industry , 
 Figure 6A has internal signs of sharp edges suggestive of cutting and pasting. *** Inverted, contrast enhanced ![file] *** Contrast enhanced ![file] *** Original ![file] *** Closeup with Forensically tool illustrates other potential problem areas ![file] , 
 These figures were elaborated by me personally and I remember quite well how I made them. Definitely, the images shown in this figure were cut and pasted from the originals in order to elaborate the figures, the overlapping was done  manually using GIMP and, also, in some cases, brightness and contrast were adjusted. I think I have the originals in several DVDs in my office; however, I m away until next Monday (January 11th). Just remind me next week and I ll be glad to send the original photographs directly taken by the microscope. It will involve some work because I think I have hundreds of photographs and I selected just a few of them to elaborate the figures that appear in the paper. This article involves work done roughly from 2004 to 2009, that is, it has been a long time during which I ve had broken computers, accidentally erased hard disks and some other troubles, therefore, some information has been lost; however, the microscopy data must be somewhere in my office because the technician in charge of this service always makes copies in DVD of the files she erases. I also keep tens of notebooks where I write down results and comments of my experiments with photographs (fixed with sticky tape), originals of western blots wrapped in aluminum foil and so on. I can take pictures of all that if you think it s necessary to prove that I am not a cheat and that I do not manipulate results. On top of this, you are cordially invited to come to my lab and check that I have nothing to hide, including the behaviour of my yeast mutants. Moreover, if you want to check by yourself that the results shown in this paper are correct and reproducible I can send you my YPC3 yeast mutant (with whom I and others have been working with no trouble whatsoever for nearly 20 years) transformed with all the constructions we tested for this article (provided that you sign the corresponding MTA). Fortunately, YPC3 is alive and kicking, because it is kept at -80u00baC and I always have a YPGal plate with YPC3 cells in my fridge. The constructions (more than fifty different ones) used to transform YPC3 (and other similar mutants I have generated) are also safely kept at -20u00baC and, some of them, in bacteria stored at -80u00baC. , 
 #9 Figure 5 of first author Rocio Drake s thesis not only includes those duplicated panels spotted by *Actinopolyspora biskrensis* (blue and magenta boxes), it also includes a panel used years before in [Perez-Castineira et al 2002](http: pubpeer.com publications E508D13C9E57C1592418405681BD9D) (red box), where it seems it was describing the complementation of YPC-1 cells, and not YPD: ![file] ![file] Could Dr. Perez-Castineira clarify? , 
 Expression of Concern. [19 July 2022] .  The Editorial Office has been made aware of potential concerns surrounding the scientific validity of this paper, hence has issued an expression of concern to notify readers whilst the Editorial Office investigates. ,