REIL proteins of Arabidopsis thaliana interact in yeast-2-hybrid assays with homologs of the yeast Rlp24, Rpl24A, Rlp24B, Arx1, and Jjj1 proteins

Figures & data

Figure 1. The cytosolic maturation machinery of the 60S large ribosomal subunit from Saccharomyces cerevisiae. The flow scheme represents the current model of the cytosolic maturation machinery⁵ starting with the pre-60S subunit after export from the nucleus (top) and ending with the translational active 60S ribosomal subunit (bottom). The direct mechanistic interaction partners of the yeast Rei1 protein within the model are indicated by light gray underlay. The cytosolic maturation releases inhibitory factors, such as the Arx1/Alb1 complex, Tif6, and Nmd3, (main path on the left) and enables assembly of the ribosome stalk (side branch on the right).

Figure 2. Protein–protein interaction matrix of the cytosolic maturation machinery of the 60S large ribosomal subunit from Saccharomyces cerevisiae. Each dark gray box within the matrix indicates a physical interaction between the cytosolic maturation factors. Interactions were retrieved and consolidated from BioGRID (Version 3.2.106). The protein names, corresponding gene codes, and the total number of physically interacting proteins are listed in the first 3 rows. The rows and columns are symmetrically ordered according to the sequence of the proteins acting within the main path of the model followed by the proteins relevant for stalk assembly, namely RPP0, Mrt4, and Yvh1. The interactions of the Rei1 protein are highlighted by a framed column. The light gray underlay accentuates the direct mechanistic interaction partners of Rei1, also refer to (Fig. 1).

Figure 3. Topology of the REIL (Rei1-like) proteins from A. thaliana and of the partial proteins which were analyzed in this study by yeast-2-hybrid assays (Table 1).(A) The 404 amino acid REIL1 and the 395 amino acid REIL2 protein each contain 4 zinc finger domains and 2 additional conserved domains, provisionally named CD1 and CD2. The truncated proteins, ZF1/2-ZF3/4, contain the 4 zinc finger domains. The CD 1/2 truncations contain only the C-terminal parts with the conserved domains CD1 and CD2. The ZF3/4-CD1/2 partial proteins lack the N-terminal pair of zinc finger domains. (B) N-terminal and C-terminal alignment of REIL1 and REIL2 homologs of Brassicales species. Zinc finger domains, ZF1 and ZF2, and conserved domains, CD1 and CD2, are indicated. The color coding of the sequence logos highlights cysteine and histidine residues, aromatic amino acids, amide, acidic, basic residues, and the conserved proline residues of the C-terminus. Color coding is given with the insert. Sequence logos were generated at http://weblogo.berkeley.edu/logo.cgi after alignment of the REIL proteins from Arabidopsis thaliana, Arabidopsis lyrata subsp. lyrata, Eutrema parvulum, Eutrema salsugineum, Brassica rapa subsp. pekinensis, Capsella rubella, Camelina sativa, Leavenworthia alabamica, Sisymbrium irio, and Aethionema arabicum.

Table 1. Protein–protein interaction matrix of REIL1, REIL2 proteins with A. thaliana homologs of the yeast proteins, Rlp24p, Rpl24Ap, Rpl24B, Arx1p, and Jjj1p

Protein Name	REIL1	REIL2
Yeast homologs	Rei1p	Reh1p	Rpl24Ap	Rpl24Bp	Rlp24p	Arx1p	Jjj1p
Gene Code	At4g31420	At2g24500	At2g36620	At3g53020	At2g44860	At3g51800	At1g74250
Full Length
REIL1	7.9	11.9	5.1	5.1	- ^a	10.3	14.1
REIL2	2.0	16.1	-	6.6	3.3	4.5	2.2
Partial REIL1
ZF1/2-ZF3/4^b	14.6	35.0	5.6	3.1	18.5	5.0	9.5
ZF3/4-CD1/2	6.6	2.5	4.0	3.9	5.9	4.5	6.5
CD1/2	5.4	8.9	3.1	5.4	5.5	3.9	9.5
Partial REIL2
ZF1/2-ZF3/4	6.2	32.8	5.9	7.7	2.9	7.4	-
ZF3/4-CD1/2	8.9	-	4.9	7.4	13.3	4.6	-
CD1/2	3.0	9.1	5.2	6.3	3.2	2.3	-

Full-length REIL bait proteins and C- or N-terminal truncations, rows also see (Fig. 3), were tested against full-length prey proteins, columns, using the yeast 2-hybrid assay. Values represent α-galactosidase activity of strains containing the respectively tested pair of heterologously expressed proteins. The activity is expressed as percent relative to a positive control strain carrying the plasmids pGBKT7–53 and pGADT7-T (Table S1)^a Few interactions tested negative and did not yield viable yeast strains (-). ^b The full and partial proteins were cloned in to the bait vector using specific primers (Table S2). The names of the truncated proteins indicate the retained zinc finger domains (ZF) and REIL-specific conserved domains (CD). The α-galactosidase activity was assayed spectrophotometrically on microtiter plates using 4–8 replications per combination and controls. The technical standard deviation of this assay was 3.5%.

Lo KY, Li Z, Bussiere C, Bresson S, Marcotte EM, Johnson AW. Defining the pathway of cytoplasmic maturation of the 60S ribosomal subunit. Mol Cell 2010; 39:196 - 208; http://dx.doi.org/10.1016/j.molcel.2010.06.018; PMID: 20670889

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