Identification of a Golgi-localized peptide reveals a minimal Golgi-targeting motif

  1. Alexandra P. Navarro
  2. Iain M. Cheeseman

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Abstract

We serendipitously identify a Golgi-localized peptide that is hiding within the mRNA for a centromere component, providing a valuable tool for the live-cell imaging of Golgi dynamics.

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  1. Version published to 10.1091/mbc.e22-03-0091
  2. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    This manuscript was evaluated at Review Commons by four individual reviewers. There was a consensus amongst reviewers that the localization behavior of altORF peptide to the Golgi is a compelling observation and that, with some additional characterization, would provide an effective cell biological tool for use in labeling and studying the Golgi. Our primary goal for this paper was to explore this surprising alternative protein hidden within the sequence of a centromere component and to establish this peptide as a cell biological tool that can be used to study the Golgi. However, the reviewers also highlighted some interesting open questions regarding the nature of this peptide. Below we summarize these core comments our current changes and plans

    • *Where within the Golgi does the peptide localize? *In the work currently included in the paper, we demonstrate that the altORF peptide robustly colocalizes with markers for the Golgi (GM130/TGN46), but not with markers for the Endoplasmic Reticulum (KDEL). However, the resolution at which we imaged the localization of the peptide was not sufficient to determine in which compartment of the Golgi the peptide resides. To address reviewer comments on the specificity of the peptide’s localization within the Golgi, we will attempt to use higher resolution imaging such as confocal or spinning disk microscopy to attempt to better resolve this.
    • *How does the peptide target to the Golgi? *In this manuscript, we show that the localization of the altORF peptide relies on a Cysteine residue present within in a minimal 10 amino acid sequence. Through treatment with 2-Bromopalmitate (2-BP; a palymityltransferase inhibitor) to disrupt its localization, our work suggests that the peptide is palmitoylated. In addition to this observation, the reviewers asked for an additional demonstration that this peptide is palmitoylated in cells. To test this, we have attempted to identify this modification using mass spectrometry of the isolated (IP) GFP tagged peptide from cells. However, we were unable to identify peptides that coincide with the modified peptide cysteine residue. Secondly, we have attempted to identify the modification using Click-chemistry labeling strategy, but this has proved to be technically challenging and infeasible. As an alternative approach for the revised version, we will attempt to perform hydroxylamine treatment followed by SDS-PAGE analysis to determine whether this results in a shift in migration of the GFP tagged altORF, as suggested by a reviewer, to provide additional evidence that the peptide is modified.
    • *Can this peptide be used to ectopically target proteins to the Golgi? *The reviewers asked whether the altORF peptide can be used to ectopically target proteins to the Golgi. In this manuscript, we demonstrate that the peptide sequence is sufficient to target both GFP and the Halo tag (two very different proteins) to the Golgi, and can be tagged at either terminus of the peptide, suggesting that it can be used as a powerful strategy to recruit other proteins to the outer surface of the Golgi. We have emphasized this point in the updated version that is included in this revision.
    • *Does this peptide alter Golgi structure? *For this peptide to provide a useful cell biological marker, it would be preferential for it not to alter cellular physiology. Our work demonstrates that expression of the altORF peptide does not affect the growth of cultured cells. For this updated version, we have performed additional analysis to test whether induced expression of the altORF peptide alters the structure of the Golgi or the localization of other Golgi-associated proteins. Based on a qualitative analysis of these cells, we do not detect any obvious changes in Golgi organization or morphology. This is now included as Supplemental Figure 2D.
      • Is this peptide expressed in human cells? *We have analyzed published ribosome profiling data that suggests that this altORF can be translated, although it is produced to a much lower degree than the full-length CENP-R protein. The short length of the peptide as well as the nature of the amino acid sequence makes this peptide highly challenging to identify via mass spec. It is also possible that this peptide would be expressed in different cell types in the human body, but not robustly expressed in cultured cells. We believe that these are beyond the scope of this paper. However, we now comment on these important points in the updated version.
      • Is this peptide “functional”? *Based on our deliberate analysis of the evolutionary conservation of this altORF within the CENPR transcript, it is clear that this peptide acquired the ability to localize to the Golgi only recently during evolution (only old world primates have this capacity). We believe that this peptide represents a great example of evolution in action, with minor sequence changes resulting in the acquisition of a new capacity and trait. However, as this peptide is not broadly conserved across mammals, it is unlikely to facilitate a core biological function that can be analyzed in cell culture. It is certainly possible that this peptide would contribute to a feature of human biology on the organismal level, but it is not feasible to test this experimentally. The functional nature of this peptide, and particularly the recent evolutionary acquisition of this novel trait are interesting points that we have now commented on in the updated manuscript (text changes in blue).
  3. Review Commons

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    Referee #4

    Evidence, reproducibility and clarity

    The manuscript reports the characterisation of a 37 amino acid alternative open reading frame (altORF) within the RNA of the centromere protein, CENP-R. The resulting peptide, when expressed in different cell lines fused to GFP, localises on the Golgi complex, exposed on the cytosolic face of Golgi membranes. It remains associated with the Golgi complex under conditions inducing fragmentation or dispersal of the Golgi complex such as mitosis and BFA. The authors identify in aa 5-14 the minimal Golgi targeting motif and in cysteine 11 a key aa for the targeting. They suppose that palmitoylation may be involved in Golgi targeting as palmitoylation inhibitors prevent its Golgi targeting. The data are clearly presented and sustain the conclusions.

    Significance

    Though the identification of a Golgi targeting motif is of potential interest, the manuscript appears to be at a preliminary stage as it fails to provide any data on the possible function of the altORF of CENP-R palmitoylation or even evidence for its existence in the cells used in the manuscript. The authors appear to be aware of the limits of their study as they conclude their study led to the identification of an "easy-to-use Golgi labeling construct". Also in this scenario, however, some key information are missing: the actual sub-Golgi localisation of the probe, its possible impact on Golgi structure and function, and the formal proof that it is palmitoylated.

    Referess cross-commenting

    I see all the reviewers agree that the manuscript has major limits. Overcoming these limits wold require years if one had to provide proofs for the existence and for the physiological relevance of this alternative ORF, and months to provide the missing information that have been highlighted by the reviewers to consider "just" the technical aspect of this altORF as a possible Golgi reporter/targeting sequence.

  4. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this study, the authors characterized a potential alternative open reading frame close to the CENP-R open reading frame that had previously been found by ribosome profiling. Its 37 amino acid peptide sequence was included in a proteomic database and is conserved in primates. Transfection of different cell lines with the GFP-tagged peptide was used for immunofluorescence and proteolytic cleavage by a cytosolic protease was used to show that it localizes to the cytoplasmic face of Golgi membranes throughout the cell cycle and Brefeldin treatment had no influence on fragmentation or reformation of the Golgi stacks. The specific localization could be confirmed using different cell lines. The use of numerous truncation mutants allowed to narrow down the minimal Golgi recognition sequence to a 10 amino acid stretch including a species-specific conserved cysteine that required palmitoylation. From these data and comparison with similar sequences in other species the authors determined a consensus sequence for this Golgi targeting sequence in primates.

    Major comments:

    1. Without ultrastructural analysis it is always difficult to judge whether a localization is limited to just one organelle. Immunofluorescence alone gives no clear answer in particular when organelles differ in size and form from cell to cell. In particular when the authors claim that the peptide may serve as a marker. For example when you are working on secretion it is important to distinguish membranes derived from ER exit sites (ERES), the ER-Golgi intermediate compartment (ERGIC), the Golgi itself and Golgi-derived vesicles. I therefore recommend to add a subcellular fractionation by which numerous fractions can be analyzed by a gel in parallel using markers for all the above mentioned different membrane origins.
    2. Is it possible to confirm the in vivo existence of this peptide? There are probably no specific antibodies available, but it should be possible to detect the peptide in enriched Golgi membrane fractions by mass spectrometry.
    3. It would be interesting to reveal the potential in vivo role of this peptide, when it exists. The authors failed to identify potential interaction partners by IP-MS, so I wonder whether its role may be different by controlling the Golgi association of other well known Golgi interactors like GM130, Golgin or GORASP proteins. Is their Golgi association altered in the presence of the peptide?
    4. Finally the authors determined a consensus sequence which they claim to be a Golgi targeting sequence, however when this is true one would expect that there are other proteins in the cell that use this consensus sequence as targeting sequence. The authors only show that the consensus is conserved among the same alternative open reading frame in primates, but to serve as a Golgi targeting sequence it should be possible to identify unrelated other proteins using this consensus by bioinformatics. What happens when an otherwise differently addressed protein is attached to this Golgi sequence, is it mislocalized?

    Minor comments:

    There are a couple of typos and smaller issues

    • In the Introduction line 2 the citation is missing and skip the "a" in line 7.
    • In the Results and Discussion section page 5, line 5 "In our ongoing work, we..."
    • In the same section close to the end in the second from the last paragraphs Figure 5B should be Figure 5C
    • In the Methods section check the temperature specifications: 4{degree sign}C or 37{degree sign}C, not 4C or 37C
    • Also in the Methods: there are no secondary antibodies recognizing complete animals (antiMouse or antiRabbit)! The antibodies are directed either against IgG or IgM (e.g. anti-Mouse IgG)
    • Some subscripts are missing: MgCl2 not MgCl2, NaN3 not NaN3
    • Also on the last page of the Methods section the antibody is specific for TGN46, not TGN146
    • Last paragraph: for concentrations use μM not uM (also in the Fig.4 legend)
    • The end of the second from the last sentence is missing.
    • In the References, is the citation for the Samandi et al. manuscript correct, just one number?
    • Legend to Suppl. Fig 3: Golgi (capital letter), (~) is missing in figure
    • Suppl. Fig1B use Courier also for peptide sequence, this will omit alignment problems

    Significance

    Overall, this study is interesting and may provide a helpful tool for cell biologists working on trafficking projects (like myself) in particular because a general Golgi targeting sequence is missing. For techniques like RUSH (Retention using specific hooks) which can be used to synchronize secretory protein traffic reliable and highly specific targeting sequences are required. I am supportive of this study, however, to be useful for the audience the authors need to provide more examples demonstrating the targeting efficiency.

  5. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    The authors have identified a alternately translated region with in the mRNA of CENP-R that encodes a small 37 aa peptide that localizes to the perinuclear Golgi region. The main premise here is the this peptide can be used as a novel Golgi marker. The peptide seems to localize peripherally to membranes and interacts with cis and TGN elements based on light microscopy. Mutational analysis indicates that a cysteine residue within a 10 aa region is critical and defines a minimal consensus sequence required for Golgi localization. Evidence is presented based on inhibition by 2-bromopalmitate that C11 is palmitoylated.

    Significance

    If this peptide probe is to be used as a Golgi-specific marker, there are several major issues that have not been addressed. The first is whether it actually binds to Golgi elements and if so, what are the specific elements? The light microscopy images are not of high enough resolution to determine if the peptide interacts with cis or TGN Golgi. The BFA experiments suggest it interacts with the TGN or some other associated vesicular compartment since staining fragments into vesicles and does not get integrated into the ER (Fig. 2B). The authors would have to use higher resolution confocal imaging or, more preferably, immuno-EM to identify exactly where the peptide is located.

    The second issue is the conclusion that the peptide is palmitoylated, which is only based on partial loss of 'Golgi' staining after 2-BP treatment (Fig. 4D). More conclusive evidence is required such as incorporation of radiolabelled or click-palmitate probes into peptide, or band shift after hydroxylamine treatment. In regard to the last point, the protein seems to migrate as a doublet on SDS-PAGE (Fig. 2D) suggesting some type of modification or cleavage that is not commented on.

    Lastly, I would be unlikely to use this as a Golgi probe for the reasons described above, as well as the fact that there is nothing known about the biological function of the peptide (this is potentially the most interesting aspect that is seemingly ignored). If you express the peptide what impact does it have on Golgi structure and function? I could envision that its binding to a Golgi element(s) could affect one of myriad functions that rely on Golgi activity.

    Referees cross-commenting

    This is more of a technical report that does not address the function of the peptide within the Golgi complex. Without this information, and identification of the compartments involved, I don't see the advantage of the probe compared to other methods. As one reviewer mentioned, this seems to be a preliminary study that is difficult to assess given the limited and ambiguous results.

  6. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    The authors note that we currently lack a robust targeting signal to direct proteins to the cytoplasmic face of Golgi membranes. The presented work clearly identifies a novel Golgi targeting sequence rich in aromatic/hydrophobic/basic residues and with a key critical cysteine (C11). One can imagine a situation where the non-cysteine residues provide an underlying affinity for cell membranes and thereby allow access to membrane-associated zDHHC S-acyltransferases. I guess the unknown question is whether Golgi specificity comes from the amino acid sequence per se (mediating specific interaction with components of Golgi membranes) or instead by specific recognition of the cysteine by Golgi-localised zDHHC enzymes. It might be worth discussing this in the paper although this should not detract from the main focus/message of the paper- the identification of a Golgi targeting peptide. Data is compelling and support the conclusions of the paper. Although much of the data is not quantified, the data provided is convincing.

    Significance

    Interesting advance for researchers in the general membrane trafficking area and S-acylation field. Provides new information that can be used to target proteins of interest to the Golgi. I note that restriction of an S-acylated peptide at the Golgi is unusual as S-acylation is usually followed by trafficking to the plasma membrane. My expertise is in S-acylation and protein trafficking

  7. Version published to 10.1101/2020.12.13.422530 on bioRxiv