Vivian Siegel

Each of the activities of signal recognition particle (SRP) is contained within a distinct domain: Analysis of biochemical mutants of SRP

Signal recognition particle (SRP), a small ribonucleoprotein required for targeting secretory proteins to the ER, has three known functions: signal recognition, elongation arrest, and translocation promotion. Because SRP is inactivated by the sulfhydryl alkylating reagent N-ethylmaleimide (NEM), we have attempted to establish structure-function relationships within SRP by assembling particles in which a single protein is modified. Alkylation of the 68/72 kd protein of SRP yields a particle that arrests elongation but fails to promote translocation and no longer interacts with SRP receptor. Alkylation of the 54 kd protein yields a particle that fails to recognize signal sequences. This approach has allowed us to map activities to specific protein domains on SRP, and should be generally useful for analyzing other ribonucleoproteins.

The affinity of signal recognition particle for presecretory proteins is dependent on nascent chain length

We have developed an assay in which incomplete preprolactin chains of varying lengths are targeted to the endoplasmic reticulum (ER) membrane in an elongation independent manner. The reaction had the same molecular requirements as nascent chain translocation across the ER membrane, namely, it was signal recognition particle (SRP) dependent, and required the nascent chain to be present as peptidyl tRNA (i.e. most likely ribosome associated) and to have its signal sequence exposed outside the ribosome. We found that the efficiency of the targeting reaction dropped dramatically as the chains grew longer than 140 amino acids in length, which probably reflected a decrease in affinity of the nascent chain‐ribosome complex for SRP. Thus at physiological SRP concentrations (10 nM) there appears a sharp cut‐off point in the ability of these chains to be targeted, while at high SRP concentrations (270 nM) all chains could be targeted. In kinetic experiments, high concentrations of SRP were found to change the time in elongation after which translocation of the nascent polypeptide could no longer occur.

Functional dissection of the signal recognition particle

Abstract Biochemical mutagenesis — alteration or removal of specific domains within a biological structure — followed by functional analysis, gives insight into structure-function relationships. We describe the analysis of the signal recognition particle, a ribonucleoprotein known to be required for the entry of most proteins into the secretory pathway, as an example of the strenght of this approach.

A second signal recognition event required for translocation into the endoplasmic reticulum

As summarized in this minireview, two different signal recognition events, one involving SRP and the other involving proteoliposomes containing the Sec61p complex, have been identified. In cotranslational protein transport, it seems that both recognition events are required for efficient translocation of the protein into the lumen of the ER. The requirement for SRP can, under certain experimental conditions, be circumvented by depletion of NAC, a heterodimeric complex that can block the tight association of nascent chain-ribosome complexes to the Sec61p complex in the ER membrane. In posttranslational protein transport, the Sec61p complex contains additional protein subunits that are required for function. It should be noted that, in all the experiments performed in which the role of SRP in cotranslational protein translocation is circumvented (Jungnickel and Rapoport, 1995; Lauring et al., 1995a, 1995b), stable translocation intermediates are allowed many minutes to establish productive interactions with the membrane. In contrast, during conditions in which the nascent chain can elongate (e.g., in vivo), the nascent chain-ribosome complex only has a brief time window during which it can initiate translocation (reviewed by Walter and Johnson, 1994). It is possible that, under these conditions, productive translocation even in the absence of NAC would require SRP. The isolation of NAC-deficient extracts that support protein synthesis will allow a test of this possibility. Finally, the role that lipids themselves may play in protein transport should not be ignored. Gierasch and coworkers (Hoyt and Gierasch, 1991, and references therein) have shown that bacterial signal peptides have an intrinsic ability to interact with lipid and that the relative ability of a mutant signal sequence to interact with lipid correlates with its function as a signal sequence in vivo. Thus, the signal sequence-discriminatory role defined by Jungnickel and Rapoport (1995) may in fact be played by lipid, with the Sec61p complex playing a necessary but nondiscriminatory role in the process. In this light, it is interesting that Martoglio et al. (1995) recently demonstrated that the signal sequence of preprolactin could be cross-linked to phospholipid. Analysis of the cross-linking efficiency of the signal sequence to phospholipid at different nascent chain lengths and with mutant signal sequences will help define the role that phospholipid plays in the process.

Recognition of a Transmembrane Domain: Another Role for the Ribosome?

In order to address the question of how the transmembrane segment induces a change in the translocon, Johnson and colleagues asked what proteins associate with the transmembrane segment during the relevant stages. Instead of using a fluorescent probe, they placed a cross-linking agent into the nascent chain (the example I shall discuss contains a cross-linking agent in the middle of the transmembrane segment). Interestingly, they found a number of proteins that could be cross-linked to the transmembrane segment (and thus are in close proximity to it) and the cross-linking pattern changed during the crucial period when the cytoplasmic and lumenal sides of the translocon seemed to be opening and closing. For example, when the transmembrane segment was two amino acids away from the tRNA, a protein of ∼41 kDa was cross-linked to the transmembrane segment. When the chain moved five amino acids further along the nascent chain pathway (and the lumenal side of the translocon closed), a new protein of ∼20 kDa now was also cross-linked to the transmembrane segment. When the transmembrane segment moved to a position that elicited the opening of the cytoplasmic side, yet another target protein of ∼10 kDa was cross-linked in addition to p20 and p41. However, none of the cross-linked proteins seemed to be integral membrane proteins since they were extracted with carbonate; furthermore, they seemed to be components of the ribosome since the same proteins were cross-linked to the transmembrane segment when the experiments were performed in the absence of ER membranes. Because the pattern of ribosomal proteins in proximity to the transmembrane segment changed during this period, it is tempting to speculate that it is these same ribosomal proteins that mediate the changes in the translocon. In contrast, none of the known components of the translocon were cross-linked to the transmembrane segment at these stages. However, since not all proteins in proximity to the transmembrane segment will be cross-linked to it, it remains possible that some unknown component of the translocon reaches into the ribosome and interacts with the transmembrane segment and is thus directly involved in triggering the changes at the translocon.These results bring up two very interesting questions. First, why does transmembrane recognition occur so early? Second, why does the translocon change from a closed–open configuration to an open–closed configuration? Although there are no answers to these questions (and it still remains to be shown that these changes in the translocon are functionally linked to the proper insertion of transmembrane domains), one thing that might be worth keeping in mind is that the lumenal and cytoplasmic sides of the ER membrane are quite distinct, both in terms of soluble factors and in terms of the reducing environment (the lumen of the ER is an oxidizing environment, allowing the formation of disulfide bonds). It is possible that the configuration of the translocon switches simply so that transmembrane domains are inserted into the ER membrane in a reducing environment. It is also possible that cytoplasmic factors may play a role in orienting membrane proteins, as suggested by the authors (seeLiao et al. 1997xLiao, S, Lin, J, Do, H, and Johnson, A.E. Cell. 1997; 90: 31–41Abstract | Full Text | Full Text PDF | PubMed | Scopus (204)See all ReferencesLiao et al. 1997references therein).Although the experiments presented in the Liao et al. paper do not quite prove the point, they do suggest the possibility that the ribosome itself plays a role in recognizing the transmembrane segment. If this is true, it is possible that the ribosome also recognizes the pause transfer sequences mentioned earlier. Since these sequences cause the cytoplasmic side of the translocon to open, it seems likely that the lumenal side becomes closed so that the permeability barrier is maintained, in a gating mechanism similar to the one described by Liao et al. 1997xLiao, S, Lin, J, Do, H, and Johnson, A.E. Cell. 1997; 90: 31–41Abstract | Full Text | Full Text PDF | PubMed | Scopus (204)See all ReferencesLiao et al. 1997.Just as we call the large and small ribosomal subunits part of the ribosome because they form an integral unit that functions as a translational machine, even though the two subunits are separate from each other in the absence of mRNA, should we also think of the ribosome as a of the translocon because it forms an integral part of the translocational machine, even though it is separate from the (other subunit of the) translocon in the absence of a translocating nascent chain? Viewed in this way, the ribosome would be the large subunit, since the mammalian ribosome is probably an order of magnitude larger than the (rest of the) translocon. Many experiments need to be done, of course, including identifying the specific factor or factors that mediate the changes in the translocon, and only time will tell whether the two subunits of the translocon communicate to mediate the response of the translocon to specific sequences within the nascent chain.

Reproducibility in research

Summary Progress in biomedical research depends in part on being able to build on the findings of other researchers – and thereby on being able to apply others’ methods to your own research. However, most of us have struggled to understand how to repeat or adapt another researcher’s study because of minimal or missing details in the Methods section of a published paper. In expensive and complex experiments involving animal models, clear descriptions of the methods are particularly important. In this and the accompanying Editorial in this issue, we discuss how crucial the Methods section is to the integrity and utility of a biomedical research paper, and encourage researchers working with animal models to follow the recently released ARRIVE guidelines when preparing their studies for publication.

Effect of Recommendations from Reviewers Suggested or Excluded by Authors

The Journal of the American Society of Nephrology (JASN) gives authors submitting original research the option of suggesting qualified reviewers or those they wish to exclude. This historical habit often leaves us wondering whether author preferences correlate with reviewer recommendations and whether differences related to reviewer selection affect decisions by editors. In a self-study presented here, we found that author-suggested reviewers, as a group, make more positive recommendations than editor-suggested reviewers (P = 0.01), although the difference disappears when recommendations are compared with those of editor-suggested reviewers of the same manuscript (P = 0.081). The distribution of recommendations by author-excluded reviewers, as a group, did not differ from those by editor-suggested reviewers; however, author-excluded reviewers impart significantly more negative recommendations than other reviewers of the same manuscript (P = 0.029). We further explored whether such differences result from individual reviewer tendencies to give generally more positive or more negative recommendations than editor-suggested reviewers and found no such tendency. Finally, editorial decisions on manuscripts reviewed by author-suggested or author-excluded reviewers do not differ from those decisions on manuscripts assigned but not reviewed by them. JASNs policy of editors making decisions independent from individual reviewer recommendations minimizes the effect of selection bias on publication decisions.

Twenty Years Already

With this issue JASN completes its 20th year of continuous publication. JASN s origin was no accident. Many members of the American Society of Nephrology had long sought to compete in the scientific and clinical marketplace of ideas and, …

I kid you not

> It is up to each of us to help all the Sarah Palins of this world, and all the Joe the Taxpayers behind them, understand and appreciate the value of all kinds of research, both within and beyond our borders As the Editor of a journal focused on the use of model organisms to advance human health

This revolution will be digitized: online tools for radical collaboration

> What if everyone in the world were in your lab – a ‘hive mind’ of sorts, but composed of countless creative intellects rather than mindless worker ants, and one in which resources, reagents and effort could be shared, along with ideas, in a manner not dictated by institutional and