In the absence of Arx1/Alb1, deletion of JJJ1 is not toxic. (A) Deletion of either ARX1 or ALB1 rescues the cold sensitivity effect of jjj1Δ strain. Ten times serial dilutions of wild-type (BY4741), jjj1Δ (ED10), arx1Δ (LMA539), alb1Δ (LMA525), arx1Δ jjj1Δ (ED52), and alb1Δ jjj1Δ (ED65) cultures were spotted on solid rich medium. Their growth on YPGlu at 23°C was compared. (B) Deletion of either ARX1 or ALB1 restores a wild-type polysome profile. The whole-cell extracts from the strains used in A were separated on a sucrose gradient as described in Figure 1B.

KAP121 overexpression has distinct effects on rei1Δ and jjj1Δ strains. (A) KAP121 overexpression does not restore the growth of jjj1Δ. Ten times serial dilutions of reiΔ (LMA523), jjj1Δ (ED10), and rei1Δ jjj1Δ (ED21) transformed with either the empty vector pFL44L or pFL44L-REI1 or pFL44L-JJJ1 or pFL44L-KAP121 were spotted on solid minimal medium without uracil at 23°C for 2 d. Generations times are indicated. (B) KAP121 overexpression does not restore nuclear localization in the absence of Jjj1. Arx1-GFP, jjj1Δ (ED49) and Arx1-GFP, rei1Δ (LMA411) strains were transformed with either the empty vector pFL44L or pFL44L-REI1 or pFL44L-JJJ1 or pFL44L-KAP121. The cells were grown in minimal media at 30°C and shifted at 23°C for 2 h. Arx1-GFP localization was observed by fluorescence microscopy.

JjjI and Rei1 are involved in the same complexes. (A) Jjj1 is associated with the 60S particles. The whole-cell extract of a strain expressing a Jjj1-TAP fusion protein (OT52) was separated by ultracentrifugation on a 10%–50% sucrose gradient. Fractions of 0.5 mL were collected. Proteins were precipitated by TCA and separated on a SDS–4%–12% polyacrylamide gel. The presence of Jjj1-TAP fusion protein was revealed by Western blotting using PAP antibodies (Sigma). (B) Jjj1 and Rei1 are present in the same pre-60S complexes. TAP purifications were performed on strains expressing Rei1-TAP, Jjj1-13myc (ED46); Jjj1-TAP, Rei1–13myc (ED19); Rlp24-TAP, Jjj1–13myc (ED87); and Rlp24-TAP, Rei1–13myc (LMA352) fusion proteins. Mak11-TAP (LMA375) and Jjj1–13myc (ED82) strains were used as control. The presence of Jjj1–13myc, Rei1–13myc, Rlp24, and Rpl1 was tested using antibodies against myc, Rlp24, and Rpl1, respectively.

Rei1 and Jjj1 are genetically linked. (A) Deletion of both JJJ1 and REI1 genes is epistatic. Ten times serial dilutions of wild-type (BY4741), rei1Δ (LMA523), jjj1Δ (ED10), and rei1Δ jjj1Δ (ED21) culture were spotted on solid rich medium. Their growth on YPGlu at 23°C was compared. Generation times are indicated. (B) REI1 overexpression partially complements the jjj1Δ growth defect. A jjj1Δ (ED10) strain (left panel) and a rei1Δ jjj1Δ (ED21) strain (right panel) were transformed with either the empty vector pFL44L or pFL44L-REI1 or pFL44L-JJJ1. Growth differences were illustrated by 10 times serial dilutions on solid minimal medium without uracil at 23°C for 2 d. Generation times are indicated. (C) REI1 overexpression partially restores 60S amount of jjj1Δ strain. jjj1Δ strain (ED10) was transformed with either pFL44L (left panel) or pFL44L-REI1 (middle panel) or pFL44L-JJJ1 (right panel). The strains were grown in minimal media without uracil at 30°C and shifted to 23°C for 2 h. The whole-cell extracts were separated by ultracentrifugation on a 10%–50% sucrose gradient. Absorbance at 254 nm was measured. Peaks corresponding to the various subunits and polysomes are indicated.

In the absence of Jjj1, Arx1/Alb1 are blocked on cytoplasmic 60S particles. (A) Arx1 and Alb1 but not Tif6 (B) accumulate into the cytoplasm in the absence of Jjj1. Arx1-GFP (LMA401), Alb1-GFP (LMA545-B), Arx1-GFP, jjj1Δ (ED49), Arx1-GFP, rei1Δ (LMA411), Alb1-GFP, jjj1Δ (ED66), Alb1-GFP, rei1Δ (LMA545-A), Tif6-TAP (ED72), Tif6-TAP, rei1Δ (ED92), and Tif6-TAP, jjj1Δ (ED89) strains were grown in minimal media at 30°C and shifted to 23°C for 2 h. The cells were observed by fluorescence microscopy. Tif6-TAP localization was observed on fixed cells using PAP antibodies (Sigma) at 1/5000 dilution followed by Cy3-conjugated secondary antibodies (Jackson Immunoresearch) at 1/250 dilution. (C) Arx1 and Alb1 are blocked on the 60S particle in the absence of Jjj1. The whole-cell extracts from the strains used in A were separated on a sucrose gradient and the fractions were analyzed as described in Figure 2A. The Western blots were performed using the fractions of the polysome profiles from 2 to 15. The presence Arx1, Tif6, and Alb1-GFP fusion proteins were checked using anti-Arx1, anti-Tif6, and anti-GFP antibodies (Santa Cruz Biotechnology) at 1/700, 1/5000 and 1/700 dilutions, respectively.

Jjj1 is involved in the 60S subunit biogenesis. (A) Two-hybrid screen identifies a physical link between Jjj1 and Rei1. (Right panel) Jjj1 was selected as a partner of Rei1 in a two-hybrid genomic screen. The domain of Jjj1 interacting with Rei1 corresponds to amino acids 162–590. The two putative C₂H₂ Zn-fingers motifs and the J domain are depicted by black and gray boxes, respectively. (Left panel) Various entire ORF were cloned into pAS2ΔΔ as bait and into pACTIIst as prey vectors, respectively. The bait and the prey vectors were transformed into the CG1945 and Y187 strains, respectively. Both strains were mated and the diploids were selected on minimal medium plates without leucine and tryptophan (−LW). The diploids that display a two-hybrid positive interaction were selected on minimal medium plates without leucine, tryptophan and histidine (−LWH). An X-Gal overlay was performed on the −LWH plate. (B) In the absence of Jjj1, the relative amount of 60S subunit is affected. A wild-type (BY4741) or a jjj1Δ strain (ED10) was grown in YPGlu and shifted at 23°C for 2 h. The whole-cell extracts from these strains were separated by ultracentrifugation on a 10%–50% sucrose gradient. Peaks corresponding to the various subunits and polysomes are indicated. Asterisks note halfmers. (C) Schematic representation of the ribosomal RNAs maturation. The relative position of the oligonucleotides used to detect specific rRNA by primer extension is indicated. (D) In the absence of Jjj1, the 27SB processing is very weakly affected. Total RNAs were extracted from culture cells of wild-type strain (BY4741), jjj1Δ (ED10), and rei1Δ mutant strains (ED70) in YPGlu medium after a shift to 23°C for 2 h. Mature and intermediate rRNA were detected either by primer extension (left panel) or by Northern blot on agarose gel (middle panel) or on acrylamide gel (right panel). The oligonucleotides used are noted. The U2 snRNA was used as a loading control.