Discussion

So far all the structural and biochemical studies on eukaryotic translation initiation involving eIF3 have been performed using natively purified protein. This approach has certain advantages

such as the presence of all possible post-translational modifications which might be of importance for the proper function of the protein in vivo. However, the fact that there are substantial amounts of eIF5 co-purified with the natively purified eIF3 makes it difficult to reliably use it in biochemical and functional experiments dealing with the GTPase activity of eIF2 (Acker et al. 2007). In vitro reconstitution of eIF3 not only overcomes the problem of co-purification of other initiation factors, but also shed light on the step-wise assembly of the complex. Moreover, step-wise reconstitution of the eIF3 complex also allows to compare different sub-complexes structurally, e.g. using electron microscopy, and define the position of different subunits in the complex. In addition, having all individual protein in hand and formation of different sub-complexes and their stable truncations increase the chance of looking at the structure of eIF3 by X-ray crystallography.

During the preparation of the grids for negative-stain EM, we noticed the low stability of the eIF3^rec complex. Dissection of the thermodynamics of the individual interactions between Tif34 with either Tif35 or Prt1 revealed the formation of a stronger complex between Tif34 and Tif35, explaining why the eIF3^rec dissociates into two stable sub-complexes. This result is in accord with the mass-spectroscopic studies on human eIF3 showing that the counterparts of Tif34 and Tif35 in human are located at the periphery of the complex, excluded from its core and tend to leave the complex more easily (Damoc et al. 2007; Zhou et al. 2008). Tif32, Nip1 and Prt1 therefore assemble the core of eIF3, forming a stable sub-complex of eIF3, which resists high concentrations of salt. This is consistent with the observation that a subcomplex of eIF3 composed of Tif32, Nip1 and Prt1 together with eIF5 is sufficient for binding to the ribosome (Valásek et al. 2003). Interestingly, despite previous reports, no interaction was observed between Prt1^181C and Tif35 at physiological salt concentrations, suggesting that Tif34 bridges the interaction between Tif32/Nip1/Prt1 and Tif34/Tif35 sub-complexes. Using limited proteolysis a stable core within the complex of Tif32/Nip1 was determined, composed of Tif32-NTD and Nip1-CTD, which withstood proteolytic digest. The same method helped us to define the last 94 residues of Prt1 and residues 14-150 of Tif35 to be responsible for their interactions with Tif34, hence forming another stable truncated sub-complex.

Figure.2.  10. limited  proteolysis  of the  sub‐complexes of  eIF3. SDS‐PAGE of the proteolysis of  Prt1^181C/Tif34/Tif35 complex (A) and Tif32/Nip1 complex (B) in the time course of one minute to one day. 

Results obtained by GluC and thermolysin are depicted in the left and right panels of each image,  respectively. (C). Analytical gel filtration profile of the Prt1^181C /Tif34/Tif35 complex digested with  thermolysin after one day. Resulted fragments do not form the complex as manifested by the presence  of two peaks shifted to the right (dark grey) compared to the non‐cleaved complex (light grey). (D). SDS‐

PAGE analysis of gel filtration run in panel C suggests the dissociation of the complex into a truncation of 

Prt1^181C and Tif34 in complex with a truncation of Tif35. Numbers correspond to different fractions of  the peak. 0 is the sample prior to gelfiltration. (E). Analytical gel filtration of Tif32/Nip1 complex treated  either with GluC for two hours (solid light grey) or with thermolysin for one day (dashed light grey). 

Comparison with the non‐cleaved complex (solid dark grey) proposes the existence of only one complex 

In vitro reconstitution of eIF3 complex allowed us in this study to fluorescently label it by attaching a fluorophore to its Tif32 subunit at position C¹⁹⁰ prior to the reconstitution. The initial results indicated 10 % efficiency of labeling. Several factors can influence the labeling efficiency including the accessibility of the targeted cystein, the pH of the reaction buffer and the nature of the fluorophore. Moreover, the more surface-exposed the cystein is, the higher is the chance of meeting and accepting the fluorophore. The fact that there is already 10% of labeling efficiency without any optimization suggests that the Cys¹⁹⁰ is to some extent exposed. The efficiency of cysteine labeling with maleimide fluorescent derivatives depends on the degree of protonation of its sulfur group, therefore an acidic medium is preferred. However, the optimal pH for high efficiency also depends of the stability of the target protein at a certain pH. In the case of Tif32*, the protein was kept at pH 7.5. Hence, further studies are needed to screen a range of pH and fluorophores for a compromise between the stability of the protein and its labeling efficiency. Our initial results indicated the power of Tif32-labeling in looking at the kinetics of interactions within eIF3 as well as eIF3 and ribosome. Binding of labeled Tif32* to unlabeled Nip1 was studied by means of stopped-flow method. The binding scheme fits to a two exponential model with an apparent binding constant of 1.83 s^-1 for the main phase. The complex of Tif32^*/Prt1/Nip1/Hcr1 was chased with unlabeled Tif32 in absence and presence of the small ribosomal subunit. Interestingly, only in the presence of 40S subunit decay in the measured signal was observed. This indicates that the dissociation rate constant of Tif32* from the Tif32*/Prt1/Nip1/Hcr1 complex is much lower than the dissociation of Tif32*, and possibly the whole complex, from the 40S. This may also indicate that upon binding of the complex to the 40S subunit, a structural rearrangement occurs leading to a lower affinity of Tif32* to the ribosome and/or the complex.

2.4. Materials and methods