4.1 Results (part B)
4.1.4 Ssb1 interacts with factors predominantly involved in early nuclear
Taking into account that r-proteins and biogenesis factors aggregate in the absence of Ssb, it was investigated whether also the latter ones represent interaction partners of this Hsp70. To define the total Ssb1 interactome, pulldown experiments were performed with TAP-tagged Ssb1 (PUIG et al., 2001). Ssb1-TAP was chromosomally expressed in yeast cells (Fig. 33A) which does not influence growth (Fig. 33B) and TAP-pulldown experiments show co-elution of several proteins (Fig. 33C). Wild type cells without any TAP-tagged protein served as negative control. Three independent experiments were performed and co-eluted proteins were identified by MS (list of hits see appendix I). The protein hits obtained were further analyzed and functionally/spatially grouped:
out of a total of 207 identified proteins (appendix I.I) 39 % represent r-proteins (23 % large SU, 16 % small SU), 9 % are involved in translation and 4 % are chaperones or cofactors of Ssb like Zuo1, Ssz1 or See1/2 (Fig. 33D).
Remarkably, factors involved in ribogenesis are clearly enriched (17 %), suggesting a specific interaction of Ssb1 with this kind of proteins. The remaining proteins with nuclear localization are indicated (11 %), all other proteins are grouped as "others" (20 %) and might represent mature interaction partners of Ssb or nascent polypeptides that co-elute with ribosome-bound Ssb. Further analysis of the 35 ribosome biogenesis factors revealed a predominant interaction of Ssb1 with early factors (18 of 35) involved in biogenesis of the 90S pre-ribosome (GRANDI et al., 2002) or the small subunit processome complex (SSU; BERNSTEIN et al., 2004) (e.g. Nop56, Nop58, or Mpp10; Fig. 33E). Most of the biogenesis factors detected (24 of 35) are localized in the nucleolus. Interestingly, almost one half of the ribogenesis factors identified (e.g. Nog2, Nop2, Nop13, Nop56, or Mpp10) are already known to become aggregation-prone in ssb1,2! cells (KOPLIN et al., 2010; WILLMUND
et al., 2013), which indicates that these factors might need the chaperoning function of Ssb.
! Figure 33: Ssb interacts with factors involved in ribosomal function, architecture and synthesis. A) Schematic overview of the experimental workflow. Cells expressing chromosomally TAP-tagged Ssb1 were grown to early exponential phase, lysed and used for TAP-pulldown (PD) experiments. Proteins which co-eluted with Ssb1 were identified via MS.
Wt cells without any TAP-tagged protein served as control. B) Growth analysis of chromosomally TAP-tagged Ssb1 in comparison to wt and knockout strains. Exponentially growing cells were adjusted to OD600 = 0.4, spotted in fivefold serial dilutions on YPD plates and incubated for 2 d at 30 °C. C) Final elution of TAP-PD from wt (no TAP) and Ssb1-TAP lysates, analyzed via SDS-PAGE followed by Coomassie staining (top) or immunological detection of distinct proteins (bottom). CBD antibody detects the remaining calmodulin-binding domain of the cleaved TAP-tag. D) Grouped protein hits after MS analysis of proteins co-eluted with Ssb1. Ribosome biogenesis factors are highlighted in red; percentage of each group is indicated. “Nuclear group” represents proteins besides ribosome biogenesis factors that localize to the nucleus,
“others” summarizes remaining protein hits. E) Further analysis of ribogenesis factors that were identified in the Ssb1 interactome as depicted in D). 90S & SSU: factors involved in 90S or small subunit processome complex biogenesis.
F) Ssb1 interactome in the presence or absence of its cofactor RAC. Ssb1-TAP PD was performed as depicted in A) in wt or RAC! (ssz1!zuo1!) cells. MS hits result from three independent experiments; total number of hits and overlap between the +/- RAC Ssb1 interactome are indicated (left); hits are grouped according to D) (right). G) Schematic overview of TAP-PD experiments to identify the nuclear and cytosolic Ssb1 interactome. H) Final elution of Ssb1-TAP from cytosolic (C) and nuclear (N) fraction, analyzed by SDS-PAGE and Coomassie staining or immunoblot. The asterisk marks untagged Ssb2 protein that is detected by the Ssb antibody. Pgk1 serves as cytosolic, Nop2 as nuclear marker. I) Analysis of MS hits of the nuclear and cytosolic Ssb1 interactome as described in F).
Results (B)
" ),"
"
*-"
In the next step, the Ssb1 interactome was examined in the absence of RAC (ssz1!,zuo1!), which is known to regulate substrate interaction and specificity of Ssb in the context of the ribosome (HUANG et al., 2005;
WILLMUND et al., 2013). In general, the number of proteins co-eluting with Ssb was reduced by about 50 % (121 hits; appendix I.II) if RAC was absent (Fig. 33F, left), but both interactome sets largely overlap (104).
Proteins of all functional groups, as defined above, are affected indicating a general reduced Ssb1-interactome in RAC! cells (Fig. 33F, right). Remarkably, the number of r-proteins is only mildly reduced, demonstrating the possibility of a RAC-independent Ssb1 interaction with theses proteins. This agrees with published data, showing a RAC-independent interaction of Ssb with mature ribosomes (GAUTSCHI et al., 2002). Whether the protein hits obtained represent direct Ssb1 interactions or display the pulldown of ribosome-bound Ssb is not distinguishable by this approach, but the reduced number of factors involved in translation hints to a real substrate binding of Ssb and r-proteins. Furthermore, the total number of co-eluted proteins and specifically that of r-proteins is reduced if ATP is present in the Ssb1 pulldown, which triggers lid opening, transition to the low affinity state and substrate release (Fig. 34). This suggests real substrate binding of at least some of the identified proteins, as the interaction of Ssb with ribosomes as a whole is not sensitive to ATP treatment (PFUND et al., 1998). In contrast to the rather unaffected r-protein interaction of Ssb1 in the absence of RAC, the number of ribogenesis factors is reduced by half (18; Fig. 33F) and some factors (10) overlap with the total Ssb1 interactome, whereas some hits (8) were only detectable in the absence of RAC.
Figure 34: The interaction of Ssb1-TAP with ribosomal and other proteins is ATP-dependent. A total lysate of Ssb1-TAP cells was divided into two parts and incubated with either apyrase to stabilize the substrate interaction of Ssb or with ATP to induce substrate release. Upon TAP-PD, final elutions were analyzed via SDS-PAGE and Coomassie staining or immunoblotting. Calmodulin-binding domain (CBD) is the remaining part of the TAP-tag, the asterisk marks Ssb2 which is detected also by the Ssb antibody; Pgk1 served as loading control.
Results (B)
" *$"
To distinguish between protein hits that represent Ssb1 substrates or interaction partners and those which just co-purified with ribosome-bound Ssb, the protocol was further adapted as follows (Fig. 33G): Ssb1-TAP cells were spheroblasted and nuclei were separated from the cytosol by differential centrifugation followed by TAP-pulldown and MS analysis of the nuclear and cytosolic Ssb1 interactome (Fig. 33H). Likely because of the longer experimental procedure less protein hits could be identified for both, the cytosolic (80 hits; appendix I.IV) as well as the nuclear (125 hits; appendix I.III) Ssb1 interactome. Interestingly, almost all cytosolic hits were also detectable in the nuclear set (71; Fig. 33I, left). Remarkably, both cytosolic and nuclear Ssb1 seems to interact with almost all r-proteins whereas ribosome biogenesis factors could only be detected in the nuclear interactome (13; e.g. Nog1, Nog2, Nop56, or Lsg1; Fig. 33I right). This suggests an interaction of Ssb with these proteins after their translation and probably no necessity of Ssb in de novo folding of ribogenesis factors.
Furthermore, RAC or Sse1 co-chaperones were present in the cytosolic as well as in the nuclear data set, suggesting a functional cooperation with Ssb in both compartments. It should be noted that most of the translation-involved factors identified in the nuclear Ssb1 interactome (7) play additional roles within this compartment or transiently localize to the nucleus, e.g. during nuclear mRNA export (e.g. Nop3, Ded1, or Tef1) (LEE et al., 1996; MURTHI et al., 2010; SENISSAR et al., 2014). Moreover, most of the proteins of the nuclear
"other" group (21) are organelle (endoplasmic reticulum / mitochondria) or membrane proteins that likely co-sedimented with nuclei (e.g. Pma1, Sec53, or Phb1).
These results provide important insights into the Ssb1 interactome, both in the cytosol and in the nucleus. Ssb1 predominantly interacts with diverse factors involved in ribosomal function, architecture and synthesis. Deletion of RAC broadly reduces the number of Ssb1 interacting proteins, among them several ribogenesis factors, but interaction with r-proteins is rather unaffected. The interaction between Ssb and r-proteins seems to be prominent and quite stable considering that almost all r-proteins could be detected under all conditions tested, even in the nuclear Ssb1 interactome. Interestingly, the few r-proteins (Rps31, Rpl40A and Rpl40B) that are synthesized as ubiquitin-fusion proteins for stabilization (FINLEY et al., 1989; LACOMBE et al., 2009) are not among the Ssb1 interactome. In addition, these proteins do not aggregate in the absence of Ssb (KOPLIN et al., 2010) suggesting that they do not need chaperone assistance by Ssb in contrast to unmodified r-proteins. This strengthens also the view that r-proteins within the Ssb1 interactome represent direct interaction partners.
Interestingly, 27 of the 59 Ssb1 interacting ribosome biogenesis factors were already identified in the ssb1,2!
aggregates (KOPLIN et al., 2010). These factors control multiple steps of nuclear and cytosolic maturation of various ribosomal precursors, suggesting a rather broad role of Ssb in ribosome biogenesis.