Functional conservation of Dhh1p, a cytoplasmic DExD/H-box protein present in large complexes

Abstract

The DHH1 gene in the yeast Saccharomyces cerevisiae encodes a putative RNA helicase of remarkable sequence similarity to several other DExD/H-box proteins, including Xp54 in Xenopus laevis and Ste13p in Schizosaccharomyces pombe. We show here that over-expression of Xp54, an integral component of the stored messenger ribonucleoprotein (mRNP) particles, can rescue the loss of Dhh1p in yeast. Localization and sedimentation studies showed that Dhh1p exists predominantly in the cytoplasm and is present in large complexes whose sizes appear to vary according to the growth stage of the cell culture. In addition, deletion of dhh1, when placed in conjunction with the mutant dbp5 and ded1 alleles, resulted in a synergistically lethal effect, suggesting that Dhh1p may have a role in mRNA export and translation. Finally, similar to Ste13p, Dhh1p is required for sporulation in the budding yeast. Taken together, our data provide evidence that the functions of Dhh1p are conserved through evolution.

Figure 1

Over-expression of Xp54 complements dhh1Δ. (A) Growth phenotypes of the dhh1Δ strain. Cells were grown to mid-log phase, serially diluted and spotted on three plates, which were then separately incubated at 16, 30 and 37°C. WT, wild-type strain; dhh1Δ, the dhh1 deletion strain; DHH1, dhh1Δ strain transformed with a plasmid-borne DHH1 allele; DHH1–HA, dhh1Δ strain transformed with a plasmid-borne DHH1–HA allele; DHH1–PA, dhh1Δ strain transformed with a plasmid-borne DHH1–PA allele. (B) Over-expression of Xp54 rescues the Ts^– phenotype of the dhh1Δ strain. DBP5, dhh1Δ strain transformed with a DBP5 gene carried on a CEN plasmid; Xp54, dhh1Δ strain transformed with an Xp54 gene carried on a CEN plasmid; 2-µm Xp54, dhh1Δ strain transformed with an Xp54 gene carried on a 2 µm plasmid; 2-µm DED1, dhh1Δ strain transformed with a DED1 gene carried on a 2 µm plasmid. (C) Abnormal cellular morphology of the dhh1Δ cells. Wild-type (WT) and dhh1Δ strains were grown in liquid YPD medium to mid-log phase at 30°C. Cells were then imaged by light microscopy using Nomaski optics.

Figure 2

Dhh1p is present in several large complexes with different sizes at different cell culture stages. DHH1–PA strain grown in liquid cultures at 30°C to (A) early-log phase (OD₆₀₀ = 0.4), (B) mid-log phase (OD₆₀₀ = 0.8), (C) late-log phase (OD₆₀₀ = 2.5) or (D) stationary phase (OD₆₀₀ = 4.8; 4 days after inoculation). Protein extracts were sedimented through 10–30% glycerol gradients and fractionated from top to the bottom of the gradient. Proteins in each fraction were further separated by SDS–PAGE and blotted for immuno-detection of Dhh1p–PA (see Materials and Methods). The positions of the protein size markers (kDa) are indicated.

Figure 3

Dhh1p is present in large complexes that sediment between 40S and 80S. The DHH1–PA strain was grown to early log phase (OD₆₀₀ = 0.4). Protein extract was loaded onto a 7–37% sucrose gradient for immunoblot analysis as described in Figure 2. The positions for the protein size markers (kDa), 40S and 60S ribosomal subunits and the 80S ribosome are indicated.

Figure 4

Dhh1p is localized in distinct cytoplasmic foci. Strain YTC335 expressing a functional Dhh1p–GFP was grown in synthetic medium lacking tryptophan and adenine to (C) 0.4 OD₆₀₀, (D) 0.8 OD₆₀₀, (E) 2.5 OD₆₀₀ and (F) saturation (4 days after inoculation). Cells were harvested, stained with DAPI and imaged by light microscopy. Localization of (A) Dbp5p–GFP and (B) Ded1p–GFP were used as controls for imaging techniques.