Dominant mutations in the late 40S biogenesis factor Ltv1 affect cytoplasmic maturation of the small ribosomal subunit in Saccharomyces cerevisiae

Abstract

In eukaryotes, 40S and 60S ribosomal subunits are assembled in the nucleus from rRNAs and ribosomal proteins, exported as premature complexes, and processed in final maturation steps in the cytoplasm. Ltv1 is a conserved 40S ribosome biogenesis factor that interacts with pre-40S complexes in vivo and is proposed to function in yeast in nuclear export. Cells lacking LTV1 grow slowly and are significantly impaired in mature 40S subunit production. Here we show that mutation or deletion of a putative nuclear export sequence in LTV1 is strongly dominant negative, but the protein does not accumulate in the nucleus, as expected for a mutation affecting export. In fact, most of the mutant protein is cytoplasmic and associated with pre-40S subunits. Cells expressing mutant Ltv1 have a 40S biogenesis defect, accumulate 20S rRNA in the cytoplasm as detected by FISH, and retain the late-acting biogenesis factor Tsr1 in the cytoplasm. Finally, overexpression of mutant Ltv1 is associated with nuclear retention of 40S subunit marker proteins, RpS2-GFP and RpS3-GFP. We suggest that the proximal consequence of these LTV1 mutations is inhibition of the cytoplasmic maturation of 40S subunits and that nuclear retention of pre-40S subunits is a downstream consequence of the failure to release and recycle critical factors back to the nucleus.

Figure 1.—

Mutant Ltv1 protein is dominant negative. (A) Sequence of Ltv1 showing NES consensus and mutations. Φ indicates Leu, Ile, Val, Met, or Phe; x indicates any amino acid. Leucine to alanine substitutions are underlined; deleted residues are indicated by an extended line. (B) Serial dilutions (4×) of exponentially growing cultures of strain LY134 (wild type) containing empty vector (pUG23), Ld57 (P_MET–ltv1_Δ169-176–GFP), or Ld58 (P_MET–ltv1_3L→3A–GFP) were spotted onto Sc–Leu (134 μm methionine) plates and incubated at 30° for 40 hr. (C) Serial dilutions (4×) of cultures of LY134 transformed with Ld73 (P_GAL–LTV1), Ld65 (P_GAL–ltv1_Δ169-176), or Ld74 (P_GAL–ltv1_3L→3A). All cultures were grown into log phase in synthetic complete media lacking leucine (Sc–Leu) with 2% glucose and then spotted onto Sc–Leu with glucose or Sc–Leu with galactose plates and incubated at 30° for 30 hr. (D) Serial dilutions (4×) of exponentially growing cultures of LY136 (Δltv1) transformed with empty vector (pRS415-Gal), Ld65 (P_GAL–ltv1_Δ169-176), or Ld74 (P_GAL–ltv1_3L→3A). Cells were grown as in C and incubated at 30° for 40 hr.

Figure 2.—

Mutant Ltv1 protein does not accumulate in the nucleus. Cells expressing nucleolar protein Sik1–RFP (LY177) were transformed with Ld47 (P_MET–LTV1–GFP), Ld57 (P_MET–ltv1_Δ169-176–GFP or Ld58 (P_MET–ltv1_3L→3A–GFP) and grown in synthetic complete media lacking histidine (134 μm methionine) into log phase (OD₆₆₀ = 0.4). Cells were imaged under both FITC and rhodamine channels. Image level was adjusted and pseudo-colored in Adobe Photoshop CS4.

Figure 3.—

Overexpression of dominant mutant Ltv1 reduces both RpS2 and RpS3 export. LY134 (wild-type) cells were cotransformed with either pRS316–RPS2–eGFP (shaded) or P_RPS3–RPS3–GFP (solid) and Ld73 (P_GAL–LTV1), Ld65 (P_GAL–ltv1_Δ169-176) or Ld74 (P_GAL–ltv1_3L→3A). Each transformation was performed twice, and cells from separate transformations were scored independently. In both experiments, all cultures were grown to stationary phase in media containing 2% glucose and then diluted into selective media with 2% galactose and incubated overnight into early log phase (OD₆₆₀ of 0.1–0.2). Live cells on poly-l-lysine-coated slides were imaged using FITC filters. Cells (n > 30) were scored for the localization of GFP fluorescence primarily to the nucleus (N > C), primarily to the cytoplasm (N < C), or equally distributed in both (N = C), and the percentage of total cells with phenotypes N > C and N = C are represented on the y-axis.

Figure 4.—

Dominant-negative mutant Ltv1 is incorporated into subunits and causes a 40S deficit. LY134 (wild-type) cells were cotransformed with empty vector (pUG23), Ld47 (P_MET–LTV1–GFP), or Ld57 (P_MET–ltv1_Δ169-176–GFP). Extracts were prepared and sedimented through sucrose density gradients as described in materials and methods. The presence of wild-type or mutant Ltv1–GFP and Rps3 in individual fractions was determined by Western blotting using anti-GFP and anti-Rps3 antibodies, respectively. Fractions containing 80S and deeper fractions were combined as indicated.

Figure 5.—

Overexpression of dominant mutant Ltv1p results in a redistribution of 20S RNA to the cytoplasm. (A) FISH detection of 20S. Wild-type cells (LY134) were transformed with plasmids Ld73 (P_GAL–LTV1) or Ld74 (P_GAL–ltv1_3L→3A). Cultures were grown to stationary phase in selective media containing 2% glucose, diluted into fresh media with 2% glucose or 2% galactose, and then grown overnight before harvesting and fixation. A Cy3-labeled DNA probe complementary to the 5′-ITS1 sequence, an indicator for 20S rRNA, was hybridized with permeabilized cells prior to imaging using TRITC filters. (B) Northern blotting for 20S pre-rRNA. Wild-type (LY134) cells containing Ld73 (P_GAL–LTV1) or Ld74 (P_GAL–ltv1_3L→3A) were cultured in the presence of glucose (Glu) or galactose (Gal) to induce expression of the plasmid borne alleles of LTV1. Total RNA was prepared from these cultures as well as from ltv1Δ (LY136) and its isogenic wild-type (LY134) and xrn1Δ (RDKY1977) and its isogenic wild-type CH1305. Top: 10 μg of total RNA was separated on a 1.5% agarose–formaldehyde gel. Northern blot was hybridized with an oligonucleotide probe specific for the 5′-end of ITS1 that detects 20S pre-rRNA and the free ITS1 fragment post cleavage. A probe specific for SCR1 was used as an internal control. Bottom: RNA samples loaded in upper panel (1 μg each) were separated on 1% agarose gel and visualized with ethidium bromide.

Figure 6.—

Overexpression of mutant ltv1 affects nuclear recycling of late 40S biogenesis factor Tsr1. (A) Cells expressing Tsr1–GFP (LY247) were transformed with vector (p415Gal), Ld73 (P_GAL–LTV1), or Ld74 (P_GAL–ltv1_3L→3A). All cultures were grown at 30° to log phase in selective media with 2% glucose and then diluted into selective media with 2% galactose and regrown for 6 hr before harvesting and imaging. (B) A leptomycin B (LMB)-sensitive strain (LY251) was cotransformed with pRS316Rio2–GFP and vector only (p415Gal), Ld73 (P_GAL–LTV1), or Ld74 (P_GAL–ltv1_3L→3A). Cells were grown in selective media with glucose and then diluted into selective media with galactose and grown overnight (16 hr) to log phase. Cells treated with LMB (+LMB) were incubated with 100 ng/μl LMB at room temperature for 20 min prior to viewing.