LSG1 dominant-negative mutants. (A) Growth of LSG1 dominant-negative mutants. CH1305 (wild-type) transformants containing empty vector (pRS425), pAJ879 (GAL10∷LSG1) or LSG1 dominant mutants (LSG1(K349T) (pAJ1109), LSG1(I204T) (pAJ1132) or LSG1(N173Y, L176S) (pAJ1131)) under control of the GAL10 promoter were streaked onto selective plates containing either glucose (repressing) or galactose (inducing) as the carbon source. Plates were incubated at 30°C for 5 days. (B) Diagram depicting positions of dominant-negative mutations within LSG1. Predicted functional motifs are demarcated. The designation of the putative G5 motif is tentative due to the degeneracy of G5 residues among G-proteins and varies from that published previously (). Arrowheads indicate positions of point mutations in dominant-negative mutants. Dominant-negative alleles are as follows: LSG1-20(Q536R), LSG1-5(T179A,L591F), LSG1-7(S107T,S351F,N406S), LSG1-10 (K349T), LSG1-14(S350P), LSG1-30(I204T), LSG1-41(K349N), LSG1-51(R267G), LSG1-52(N173Y,L176S) and LSG1-53(K349R). ^*, ^**, ^*** denote multiple mutations in LSG1-7, LSG1-52 and LSG1-5, respectively. For polysomes in (C), corresponding cultures in (A) were grown to saturation, diluted to OD₆₀₀∼0.1 in fresh raffinose-containing medium and incubated to OD₆₀₀∼0.3. All cultures were then induced with galactose for 4 h, treated with cycloheximide and extracts run on 7–47% linear sucrose gradients as described in Materials and methods.

Nuclear accumulation of Rpl25-eGFP in lsg1 and rpl10 mutants. (A) Rpl25-eGFP (pAJ908) was continuously expressed in wild-type cells (CH1305) carrying empty vector (pRS425), pAJ879 (GAL10∷LSG1), pAJ1109 (GAL10∷LSG1[K349T]) or pAJ1132 (GAL10∷LSG1[I204T]). Localization after growth in raffinose was compared to that after a 3 h induction with galactose. Rpl25-eGFP (pAJ907) was visualized in the rpl10[G161D] mutant (AJY1657) at semipermissive temperature (B) and in GAL∷RPL10 cells (DEH221+) with or without 4 h of repression by glucose (C). Cells were prepared for visualization as described in Materials and methods.

lsg1 and rpl10 mutants trap Nmd3-GFP on cytoplasmic 60S. Localization of chromosomally expressed Nmd3-GFP in the presence of LMB. (A) Nmd3-GFP was visualized in strain AJY1705 (NMD3-GFP∷KanMX6 CRM1[T539C]) carrying empty vector (pRS425), pAJ879 or pAJ1109. Cells were induced with galactose or maintained in glucose for 3 h before the addition of LMB. (B) RPL10 expression was maintained in AJY1836 (GAL1-10∷RPL10 NMD3-GFP CRM1[T539C]) in galactose or repressed in glucose before the addition of LMB and visualization of Nmd3-GFP. In (C), Nmd3AAA-GFP (pAJ754) was visualized in strain DEH221+ (GAL1-10∷RPL10) after RPL10 repression or in the rpl10[G161D] mutant AJY1657 cultured at 25°C. (D) Cells were cultured as in , extracts prepared and fractionated by sucrose gradient sedimentation. Western blotting of proteins in each fraction was carried out using α-Nmd3p or α-Rpl1ap.

Genetic interactions among LSG1, NMD3 and RPL10. (A) lsg1 and nmd3 mutants exhibit synthetic lethality. AJY1513 (nmd3–3), AJY1518 (nmd3–4), AJY1511 (lsg1∷KanMX4/pAJ626[LSG1 URA3]), AJY1512 (lsg1∷KanMX4nmd3–3/pAJ626[LSG1 URA3]), AJY1521 (lsg1∷KanMX4nmd3–4/pAJ626[LSG1 URA3]) and a congenic wild-type strain were transformed with lsg1-2 and lsg1-3 mutant alleles on LEU2 plasmids. Transformants were restreaked onto 5-FOA plates to exclude the wild-type LSG1∷URA3 plasmids. Plates were incubated at 25°C for 10 days. (B) Growth suppression of a dominant-negative LSG1 allele by high-copy NMD3. In all, 10-fold serial dilutions of stationary-phase cultures were spotted onto selective plates containing galactose and incubated for 5 days at 30°C. The strains tested were: wild-type (CH1305) transformed with pAJ1109 (GAL10∷LSG1[K349T]) in combination with empty vector (pRS416), pAJ409 (NMD3, CEN), pAJ363 (NMD3, 2μ) or pAJ1143 (GAL1∷NMD3,CEN). The growth of wild-type (CH1305), containing empty vectors, and LSG1(K349T), suppressed with high-copy NMD3, is shown for comparison. (C) Growth suppression of dominant-negative LSG1 and rpl10^ts mutants by NMD3 suppressor alleles. Cultures of either CH1305 containing pAJ1278 (GAL10∷LSG1[K349T]) or AJY1657 (rpl10[G161D]), each also containing empty vector (pRS415), pAJ538 (NMD3-myc), pAJ415 (NMD3[L291F]-myc) or pAJ1315 (NMD3[I112T, I362T]-myc), were diluted and plated as for (B). The CH1305 plate was incubated 5 days at 30°C and the AJY1657 plate for 3 days at 35°C.

Ectopic expression of wild-type NMD3 and introduction of suppressor mutations allows Nmd3-GFP to recycle in the presence of lsg1 and rpl10 mutants. (A) Cultures of strain AJY1705 (NMD3-GFP CRM1[T539C]) containing pAJ363 (NMD3, 2μ) and either empty vector (pRS425), pAJ879 (GAL10∷LSG1) or pAJ1109 (GAL10∷LSG1[K349T]) were maintained in raffinose or galactose was added to induce the LSG1 alleles. Cells were then treated with LMB, fixed, DAPI stained and visualized as described in Materials and methods. For (B), AJY1657 (rpl10[G161D]) containing either pAJ1069 (NMD3[L291F]AAA-GFP) or pAJ1288 (NMD3[I112T, I362T]AAA-GFP) was grown constitutively at 25°C and treated as in (A). DEH221+ containing pAJ1069 (NMD3[L291F]AAA-GFP) was cultured and prepared for microscopy as described for . (C) Overnight cultures of strain AJY1896 (nmd3∷TRP1 CRM1[T539C]) containing pAJ582 (NMD3-GFP) or pAJ1287 (NMD3[I112T, I362T]-GFP) as the sole copies of NMD3 and either empty vector (pRS426), pAJ1312 (GAL10∷LSG1) or pAJ1278 (GAL10∷LSG1[K349T]) in medium containing raffinose were diluted two-fold in fresh medium in the presence of either raffinose or galactose to induce LSG1 expression. After 3 h, cells were fixed and treated for visualization as in (A).

Nmd3 suppressor proteins have lower affinity for 60S subunits. (A) Three-fold increasing amounts (1 × ∼30 ng) of affinity-purified GST-Nmd3p wild-type, [I112T, I362T] suppressor, [L291F] suppressor or [V340D] loss of function mutant proteins were incubated alone or with purified 60S subunits and run on 2.5% acrylamide/0.5% agarose composite gels as described in Materials and methods. The position of Nmd3p and 60S subunits was determined by Western blotting using anti-GST and ethidium bromide staining, respectively. (B) Cultures of strain W303 containing empty vector (pRS425), pAJ538 (NMD3-myc), pAJ1315 (NMD3[I112T, I362T]-myc), pAJ1070 (NMD3[L291F]-myc) or pAJ1299 (nmd3[V340D]-myc) were collected and α-myc immunoprecipitations performed as described in Materials and methods. Samples and a purified 60S control were run on a 12% SDS–PAGE gel and analyzed by Western blotting using α-myc (Nmd3p) or α-Rpl8p. The gel was stained with Coomassie Blue. (C) Cultures of strains W303 or AJY1657 transformed with either pAJ538 (NMD3-myc) or pAJ1315 (NMD3[I112T, I362T]-myc) were grown constitutively at 30°C (W303) or shifted to 37°C for 3 h (AJY1657) before harvesting. Extracts were prepared in the presence of cycloheximide followed by analysis on sucrose gradients as described in Materials and methods. Sucrose gradient fractions were analyzed by Western blotting using α-myc (Nmd3p) or α-Rpl12p. Densitometric traces of α-myc blots were conducted using NIH Image V.1.62.

NMD3 suppresses ribosome biogenesis/export defects in lsg1 and rpl10 mutants. (A) Wild-type strain CH1305 was cotransformed with the various combinations of vectors: two empty vectors (pRS425) and (pRS426); pRS425 and pAJ363 (NMD3); pAJ879 (GAL10∷LSG1) and pRS426; pAJ879 and pAJ363; pAJ1109 (GAL10∷LSG1[K349T]) and pRS426; or pAJ1109 and pAJ363. Cells were grown in the presence of galactose for 3 h and prepared for microscopy as described in Materials and methods. (B) DEH221+ (GAL1-10∷RPL10) cells containing pASZ11-RPL25-eGFP (RPL25-eGFP) with either pRS315 (empty vector), pAJ410 (NMD3 2μ), pAJ123 (NMD3 CEN) or pAJ415 (NMD3[L291F] CEN) were grown in galactose-containing medium and diluted four-fold into glucose-containing medium to repress RPL10 expression. After 4 h, cells were prepared for visualization as above. Similar results were obtained with NMD3(I112T, I362T) (data not shown). (C) Cultures of AJY1657 (rpl10[G161D]) cells carrying empty vector (pRS425), pAJ538 (NMD3-myc) or pAJ1315 (NMD3[I112T, I362T]-myc) were shifted to 37°C for 3 h before harvesting. Extracts were prepared in the presence of cycloheximide followed by analysis on sucrose gradients as described in Materials and methods. (D) CH1305 cells carrying pAJ1109 (GAL10∷LSG1[K349T]) with either empty vector (pRS426) or high-copy pAJ363 (NMD3) were cultured in raffinose media followed by induction with galactose. After 4 h, cells were collected, extracts prepared and gradients run as in (C). Arrows indicate increased 60S levels and polysomes in cells coexpressing Lsg1p dominant-negative alleles and high-copy Nmd3p.

Model explaining the effects of Lsg1p and Rpl10p on Nmd3p shuttling and 60S export. (A) The interactions between Rpl10p, Nmd3p and Lsg1p are shown under normal cellular conditions. (B) The consequence of failing to recycle Nmd3p due to defects in Lsg1p or Rpl10p. Both models are described in the Discussion.