Figure 42: Ribosome-bound NAC enhances puromycin induced detachment of ribosomes from microsomes. Native rough microsomes were treated with or without puromycin in the presence or absence of recombinant wt NAC protein or the ribosome-binding mutant NAC_RRK-AAA. Microsomes were pelleted and supernatant and pellet fraction were analyzed via Western blot and detection of Sec61 alpha (marker of microsomes) or Rpl10 (ribosomal marker).
5.4 Summary, discussion and outlook
In this part of the thesis some important in vitro experiments were performed that strongly support comprehensive in vivo analyses investigating the role of NAC during co-translational ER targeting. It could be shown, that similar to the already described yeast NAC (WEGRZYN et al., 2006) mutation of the conserved RRK29-31 sequence motif within beta-NAC to AAA residues abolishes in vitro ribosome binding also in the case of C. elegans NAC. It could be shown that ribosome binding is a critical determinant that enables NAC to negatively regulate binding of ribosomes to stripped microsomes and to induce detachment of ribosomes from native microsomes. The strong effect of NAC in ribosome detachment is remarkable, as the affinity of ribosomes for the Sec61 translocon is high (ADELMAN et al., 1973; BORGESE et al., 1974; KALIES et al., 1994; PRINZ et al., 2000). These findings are supportive and integrated into a series of in vivo experiments that were performed in collaboration. There it could be shown that NAC depletion shortens the life span of C. elegans and provokes both ER- and mitochondrial-specific stress responses. The reduced expression of NAC in adult worms results in a global but SRP-independent mistargeting of ribosomes translating non-ER substrates to the ER (Fig. 43).
Remarkably, in the absence of NAC some newly synthesized proteins containing a mitochondrial signal sequence not only get mistargeted, but also are able to override the Sec61 proofreading (JUNGNICKEL &
RAPOPORT, 1995) and to enter the ER lumen, where the mislocalized proteins are then subjected to the ERAD pathway. The SRP-dependent targeting and translocation of ER proteins, however, seems to function normally and is not affected in cells lacking NAC. This changes in contrast if NAC is overexpressed, leading to a remarkable reduction of membrane-attached ribosomes, enhanced ER stress and a shift of SRP binding to later polysomes. Thus, increased levels of NAC delays the timely targeting of RNCs to the ER membrane and specific SRP substrates are less efficiently targeted under these conditions. Taken together, these findings indicate an essential antagonistic role of NAC as a negative regulator of co-translational protein transport to the ER in combination with the positive regulator SRP. Thereby, NAC prevents unspecific binding of vacant ribosomes or RNCs translating non-ER proteins to the Sec61 translocon. Only the opposing activity of both, NAC and SRP, guarantees a robust sorting of RNCs that ensures the fidelity and specificity of protein translocation into the ER.
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A role of NAC in ER targeting has been observed or suggested by several earlier in vitro studies (WIEDMANN
et al., 1994; LAURING et al., 1995; MÖLLER et al., 1998) already more than twenty years ago. Later analyses, also performed in vitro, however, provided contrary results and challenged the earlier findings (NEUHOF et al., 1998; RADEN &GILMORE, 1998). The molecular reasons behind these discrepancies seem to be diverse as all studies applied a widely used system where in vitro translation extracts prepared from either wheat germ or rabbit reticulocyte lysates are supplemented with canine pancreas rough microsomes (BLOBEL &DOBBERSTEIN, 1975). The heterogeneity of these experiments is even increased by the addition of purified NAC protein from various sources like heterologous expression from bacteria or isolation of NAC from yeast or bovine.
Furthermore, the method of microsome preparation varied in earlier studies as well as the concentration of supplemented NAC protein. Thus, investigations that challenged the first findings of the Wiedmann group (WIEDMANN et al., 1994; LAURING et al., 1995; MÖLLER et al., 1998) used a modified method, in which microsomal membranes were stripped of their endogenous ribosomes with puromycin rather than by EDTA treatment (LAURING et al., 1995; MÖLLER et al., 1998). Furthermore, these groups used sub-physiologic NAC concentrations, which might be the reason why their data did not confirm an impact of NAC on SRP binding to ribosomes or on ribosome binding to microsomes. Although there are experimental variations, the exact basis for the inconsistency of the resulting data has remained moot. Regardless of the discrepancies these older studies significantly differ from in vitro analyses performed in this study, as they used a highly heterologous and mixed system analyzing plant or mammalian translation extracts, purified bovine NAC or recombinant NAC from bacteria and rough microsomes from canine pancreas. Experiments from the present study in contrast did only
Figure 43: A double secure mechanism sustains ER targeting specificity in vivo. In C. elegans, NAC prevents vacant ribosomes and ribosome-nascent chain complexes (RNCs) translating cytoplasmic or mitochondrial proteins from mistargeting to the ER membrane. Thereby, NAC acts as a crucial negative regulator of ER targeting by blocking the binding of ribosomes to the Sec61 translocon. SRP specifically binds to ribosomes displaying hydrophobic signal sequences what promotes binding of these RNCs to the Sec61 channel. Only the opposing activities of NAC and SRP guarantee a highly accurate targeting of newly synthesized proteins to different cellular compartments. In the absence of NAC (red arrows) all types of ribosomes get targeted to the ER membrane and mitochondrial proteins are even able to pass the Sec61 proofreading function and to enter the endoplasmic reticulum, which causes stress responses in both compartments, the ER and mitochondria (figure taken from KRAMER et al., 2015).
Discussion & Outlook (C)
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combine E. coli recombinantly expressed and purified C. elegans NAC protein with ribosomes and microsomes directly isolated from the nematode model system. Data obtained from the above described in vitro experiments are therefore reliable, especially as they perfectly fit to the comprehensive in vivo analyses that were addressed directly in the C. elegans model.
The only in vivo studies performed prior this investigation were done in yeast cells and showed no aberrant translocation phenotype upon NAC deletion (REIMANN et al., 1999; DEL ALAMO et al., 2011). Despite the fact that NAC coordinates SRP recruitment to ribosomes and modulates its substrate selection, NAC deletion does not seem to affect ER targeting specificity in yeast (DEL ALAMO et al., 2011; ZHANG et al., 2012), which might be due to several reasons. As a unicellular organism yeast is able to rapidly adapt to changing situations and to overcome the deletion of important factors. The loss of SRP for example leads to a fast adaptive response within hours including slow down of growth rates and specific changes in global gene expression (MUTKA &WALTER, 2001). In addition, yeast evolved SRP-independent, and thus likely also NAC-independent, protein targeting pathways involving Sec62, 63, 71 and 72 (DESHAIES &SCHEKMAN, 1989; ROTHBLATT et al., 1989; DESHAIES
et al., 1991; AST et al., 2013), that might substitute for failures of SRP if NAC is missing. Another reason could be the correct engagement of all Sec61 translocon pores, as yeast cells divide fast and comprehensively use the secretory pathway. Since NAC depletion in C. elegans does not influence correct SRP-targeting but causes unintended binding of ribosomes only to unoccupied translocon pores, a negative effect of NAC deletion might not be detectable in the yeast model system. Although, it is still possible that yeast NAC fulfills a similar function as C. elegans NAC, establishing a reliable in vivo prove for that will remain challenging. Unpublished data from our lab however showed that the in vitro analyses described above are reproducible in a S. cerevisiae system, emphasizing the evolutionary conservation of the NAC function.
Based on the findings in this part of the study, important open questions remain that should be addressed in the future:
(i) How are the opposing activities of NAC and SRP coordinated in a timely and precise manner on the ribosome? A competition between NAC and SRP for ribosome binding has been previously indicated by several in vitro studies (POWERS &WALTER, 1996; MÖLLER et al., 1998; ZHANG et al., 2012), and both NAC and SRP, can bind to RNCs carrying signal sequence containing or transmembrane nascent chains (DEL ALAMO et al., 2011). In vitro analyses with stable RNCs carrying nascent chains of different length and sequence could help to investigate the binding mode and potential competition of NAC and SRP. Investigating RNCs with or without targeting sequences for the ER or mitochondria in combination with a different localization of these signals within or outside the ribosomal tunnel might answer important questions about the coordination of NAC and SRP binding. As an important in vivo analysis ribosome profiling could be used to investigate the binding characteristics of NAC and SRP to RNCs on a global scale. This method not only allows identification of the nature of an RNC associated mRNA but also the exact position of ribosomes on this transcript (INGOLIA et al., 2012), which might help to understand the coordination of NAC and SRP binding.
Apart from crosslinking data suggesting that NAC interacts with several kinds of nascent chains (DEL ALAMO
et al., 2011), little is known about the substrate specificity or the substrate-binding site of NAC. Structural modeling has recently identified highly conserved hydrophobic patches (unpublished data), which could be used for site-specific crosslinking to address the binding mode of NAC and the potential substrate recognition motif.
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(ii) Where exactly does NAC bind to ribosomes? Crosslinking experiments suggested different binding sites for NAC close to the tunnel exit at uL23 (WEGRZYN et al., 2006) and/or eL31 (PECH et al., 2010; ZHANG et al., 2012), however, structural analysis of NAC bound to ribosomes are still missing. SRP and the Sec61 translocon both contact the ribosome amongst other sites via uL23 (HALIC et al., 2004; VOORHEES et al., 2014), which raises the question of simultaneous binding together with NAC. eL31 is placed like uL23 near the tunnel exit, thus, NAC could sterically inhibit the translocon interaction site also via eL31. Structural analysis via cryo-EM tomography of NAC bound to ribosomes might help to identify the exact ribosome-binding sites and understand the function of NAC as a regulator of ER targeting on a molecular basis.
(iii) How is NAC displaced from ribosomes translating ER proteins? It could be shown in this study that the presence of ribosome attached NAC prevents binding to the Sec61 translocon. This requires NAC displacement from ribosomes, however, at which specific step the inhibitory action of NAC is counteracted by the SRP pathway is unclear. It is conceivable that the interaction of SRP, of the SRP receptor or binding to the Sec61 translocon finally displaces NAC from RNCs. One likely mechanism is that displacement of NAC is mediated already by SRP that contacts one of the proposed ribosomal binding sites of NAC (WEGRZYN et al., 2006), although other data indicate simultaneous binding of both complexes (DEL ALAMO et al., 2011; ZHANG et al., 2012). The high affinity of SRP to RNCs expressing hydrophobic signal sequences or transmembrane domains (WALTER et al., 1981; NEUHOF et al., 1998) could give SRP a selective advantage over NAC for ribosome binding. Defined RNCs could be used in presence or absence of SRP and in combination with purified SR or microsomes containing functional SR and Sec61 to investigate which factors are critical for NAC detachment from ribosomes.
(iv) How can nascent mitochondrial proteins enter the ER and override the Sec61 proofreading function? In this study it could be shown that in the absence of NAC proteins not only get mistargeted to the ER but some mitochondrial proteins are even imported. Mitochondrial signal sequences are characterized by an amphipatic helix resulting from alternating positive and hydrophobic residues (NEUPERT &HERRMANN, 2007) in contrast to the pure hydrophobic signal sequence of ER proteins. Thus, it remains an open question how those proteins are able to at least partially open the Sec61 channel. Performance of an import assay using defined RNCs carrying transmembrane domains of the outer or inner membrane of mitochondria in combination with microsomes might answer the question which type of sequence is able to override the Sec61 proofreading function.
(v) How does the function of NAC in ER targeting change during aging, which is known to lead to NAC dissociation from ribosomes? Recent studies showed that NAC is sequestered by cytosolic aggregates under acute and chronic protein folding stress conditions (KIRSTEIN-MILES et al., 2013). This suggests a decline of NAC function which could also lead to targeting defects in stressed or aged cells, that are often associated with ER or mitochondrial stress. It would be interesting to investigate whether proteotoxic stress causes dysfunction of NAC and, thus, leads to impairment of protein biogenesis and transport pathways.