Function of a plant stress-induced gene, HVA22. Synthetic enhancement screen with its yeast homolog reveals its role in vesicular traffic

Abstract

Expression of the barley (Hordeum vulgare) HVA22 gene is induced by environmental stresses, such as dehydration, salinity, and extreme temperatures, and by a plant stress hormone, abscisic acid. Genes sharing high level of sequence similarities with HVA22 exist in diverse eukaryotic organisms, including animals, plants, and fungi, but not in any prokaryotic organisms. The yeast (Saccharomyces cerevisiae) HVA22 homolog, Yop1p, has been shown to interact with the GTPase-interacting protein, Yip1p. Deletion of YOP1 led to only a modest reduction of the stationary phase titer at 37C. A synthetic enhancement mutant screen was performed in the yop1 deletion background to identify genes interacting with YOP1. The open reading frame YOR165W (renamed SEY1 for synthetic enhancement of YOP1) was identified as a YOP1-dependent complementation gene. The yeast SEY1 is a homolog of the Arabidopsis RHD3 gene whose mutations cause the accumulation of transport vesicles near the tips of defective root hairs. The yeast double mutant of yop1 and sey1 is defective in vesicular traffic as evidenced by the accumulation of transport vesicles and the decrease in invertase secretion. Based on these observations, we suggest that Yop1p/HVA22 regulates vesicular traffic in stressed cells either to facilitate membrane turnover, or to decrease unnecessary secretion.

Figure 1

Comparison of yeast Yop1p, barley HVA22, human Dp1, and Arabidopsis AtHVA22d. A, Amino acid sequence alignment of Yop1p and homologs from barley, human, and Arabidopsis. Identical residues are highlighted; similar residues are outlined. The entire barley sequence is shown, and the others are truncated accordingly. B, Hydropathy plot of Yop1p and homologs from barley, human, and Arabidopsis, comparing the hydrophobic and hydrophilic regions in the four proteins.

Figure 2

The Yop1Δ mutant shows a temperature-sensitive growth phenotype. Liquid YPD media was inoculated with wild type or yop1Δ (deletion mutant) and shaken at 25°C or 37°C. The density of the cultures was quantified by measuring A₆₀₀. Values shown are averages ± se, n = 3. A, At 25°C, there is no significant difference between wild type (solid line) and the yop1Δ mutant (dashed line). B, At 37°C, the A₆₀₀ of the mutant cultures are about 75% that of wild type after 55 h.

Figure 3

sey1 synthetically enhances yop1Δ. A synthetic enhancement mutant was transformed with pRS315 containing no insert, a YOP1 genomic clone, a SEY1 genomic clone, or an N-terminal deletion mutant of YOP1 and streaked out on rich media. After 1 week, colonies were examined for sectoring.

Figure 4

Molecular nature of sey1 mutant alleles and comparison with Arabidopsis RHD3. A, Alleles 1 through 3 were cloned by the gap repair procedure described in “Materials and Methods.” Allele 4 was cloned by PCR (see “Materials and Methods” for details). Recovered clones were sequenced to identify the lesions. B, Alignment of the N-terminal region of Sey1p and RHD3. Identical residues are highlighted; similar residues are outlined. The location of the sey1 lesions are marked by stars, and the GTP-binding motifs are double underlined.

Figure 5

The Sey1Δ mutant shows no growth phenotype at 25°C or 37°C. Liquid YPD media was inoculated with wild type or sey1Δ (deletion mutant) and shaken at 25°C or 37°C for 74 h. The density of the cultures was quantified by measuring A₆₀₀. Values shown are averages ± se, n = 3. A, At 25°C, there is no significant difference between wild type (solid line) and the sey1Δ mutant (dashed line). B, At 25°C, there is no significant difference between wild type (solid line) and the sey1Δ mutant (dashed line).

Figure 6

A yop1Δ/sey1 double mutant accumulates transport vesicles. Thin-section EM of wild-type yeast (A), yop1Δ mutant (B), sey1Δ mutant (C), and a yop1Δ mutant carrying sey1-4 (D). The yop1Δ/sey1-4 mutant was used to recover white colonies at 37°C, which were grown in liquid media at 37°C, then shifted to room temperature for 3 h. n, Nucleus; V, vacuole; Bb, Berkeley bodies.

Figure 7

The synthetic enhancement mutant has a secretion defect that can be rescued by YOP1 or SEY1. A, Wild type or yop1Δ/sey1-4 mutant yeast were grown in yeast peptone (YP) supplemented with 5% (w/v) Glc at 37°C. During log phase, the cultures were washed and resuspended in YP media supplemented with 0.05% (w/v) Glc at 25°C. After 3 h, cells were collected and resuspended in 10 mm NaN₃. Samples were divided in two for external invertase assays and total invertase assays. The values were used to calculate the percentage of invertase secreted. Values shown are averages ± se, n = 3. B, The yop1Δ/sey1-4 mutant was transformed with pRS315 containing a YOP1 genomic clone, no insert, or an SEY1 genomic clone. Cultures were grown in synthetic complete-Leu media supplemented with 5% (w/v) Glc at 37°C. During log phase, the cultures were washed and resuspended in synthetic complete-Leu media supplemented with 0.05% (w/v) Glc at 25C. After 2.5 h, cells were collected and resuspended in 10 mm NaN₃ and processed as in A. Values shown are averages ± se, n = 3.

Figure 8

Yop1p interacts with itself in a two-hybrid assay. A, Yeast host strain YRG-2 transformants carrying: i, positive control plasmids pADWT and pBDWT; ii, GAL4AD-YOP1 and GAL4BD; iii, GAL4AD and GAL4BD-YOP1; or iv, GAL4AD-YOP1 and GAL4BD-YOP1 were streaked out on synthetic media lacking Leu, Trp, and His and grown at 30°C for 5 d. B, The amino acid sequence of Yop1p showing a potential Leu zipper (highlighted).

Figure 9

Summary of reported interactions involving YOP1 and SEY1. Interactions involving YOP1 and SEY1 are shown schematically and are listed as follows: 1 and 2, two-hybrid interaction (Yang et al., 1998); 3, two-hybrid interaction and glutathione S-transferase pull down (Calero et al., 2001); 4, synthetic enhancement (this study); 5, synthetic enhancement (Sapperstein et al., 1996); 6 through 9, two-hybrid interaction (Ito et al., 2001a); 10, sequence similarity (this study).