The GDI1 genes from Kluyveromyces lactis and Pichia pastoris: cloning and functional expression in Saccharomyces cerevisiae

Introduction

Vesicle targeting and fusion events in eukaryotic cells require strict regulatory mechanisms. Small GTPases of the Rab family, first identified in the yeast Saccharomyces cerevisiae (Salminen and Novick, 1987) are such regulatory factors. They cycle between the GDP-bound inactive and GTP-bound active form. The GDP dissociation inhibitors (GDIs) regulate Rab proteins by maintaining them in their inactive, soluble form in the cytosol (Garrett et al., 1994; Ullrich et al., 1993). GDI proteins are highly homologous within a wide variety of organisms including Homo sapiens, S.cerevisiae, Arabidopsis thaliana and others (Z̆árský et al., 1997). Functional similarity has been demonstrated, for example, by the ability of a cDNA coding for A. thaliana Gdi to complement the sec19-1 mutation (allelic to gdi1) of S. cerevisiae (Z̆árský et al., 1997) and by in vitro experiments with yeast Sec4p and Drosophila or bovine GDI (Garrett et al., 1993). The crystal structure of bovine GDI has been determined (Luan et al., 2000; Schalk et al., 1996). The protein has been shown to consist of two domains. Domain I ofS.cerevisiae Gdi1p binds to Rab GTPase (Luanet al., 1999; Schalk et al., 1996; Wu et al., 1998), whereas Domain II regulates membrane attachment and release of Rab (Gilbert and Burd, 2000).

In clinical studies, it has also been shown thatmutations in the human GDI1 gene coding forα-GDI lead to mental retardation (D'Adamo et al., 1998), and this emphasizes the importance ofthe role of GDI proteins as components ofcellular regulation from protein secretion inyeast to neuronal development and action in man.

In this paper we report on the complete nucleotide sequences of two genes coding for GDI proteins. The deduced amino acid sequences of the genes from Kluyveromyces lactis and Pichia pastoris show identities of 81% and 72%, respectively, when compared to S. cerevisiae Gdi1p.

Materials and methods

Results and discussion

Cloning of the Kluyveromyces lactis GDI1 gene

The K. lactis GDI1 gene was found during sequencing of a library clone obtained from a screening not related to this work. Initial testing of functionality was done by introducing the library clone into the sec19-1 mutant strain, in which it suppressed the temperature-sensitive phenotype. Full complementation at both 37°C and 38°C was also achieved with the centromeric plasmid YCpM–KlGDI1 containing the K. lactis GDI1 ORF obtained byPCR. A genomic fragment of 2794 bp was sequenced (Figure 1). It contains the ORF of 1338 bp, coding for a protein of 445 amino acidswith a calculated molecular weight of 50.3 kDaand a pI value of 5.4 (http://www.expasy.ch/cgi-bin/pi_tool). No intron was found. On the opposite strand of the promoter region, at position −511, another potential ORF starts. The partial sequence has its highest homology with the putative D-2-hydroxyacid dehydrogenase YGL185c of S. cerevisiae.

Cloning of the Pichia pastoris GDI1 gene

To obtain the P. pastoris GDI1 gene, we used the P.pastoris genomic library to transform the sec19-1 temperature-sensitive yeast strain. After the transformation, cells were plated on selective medium and incubated at 24°C for 16 h to allow gene expression to start. Plates were then shifted to the restrictive temperature of 35°C and incubated for a further 2 days. Approximately 2 million transformants were screened on 28 plates, yielding ca. 1000 temperature-resistant colonies. 144 Colonies were picked at random, patched on YPD plates and grown overnight at 35°C. The patches grown on YPD were replicated onto plates containing 5-FOA and incubated for 3 days at 35°C. Patches showing papillar growth were discarded as originating from possible revertants not needing the URA3-containing library plasmid for survival. 16 patches showing no growth were selected, and the plasmids were extracted and transformed into E. coli for preparation of DNA. Retransformation into the sec19-1 strain yielded two library plasmids that suppressed the temperature-sensitive phenotype. From both plasmids the P. pastoris GDI1 gene was identified by sequencing. Full complementation at both 37°C and 38°C was also achieved with the centromeric plasmid YCpM–PpGDI1 containing the P. pastoris GDI1 ORF amplified by PCR. In total, a genomic fragment of 2876 bp was sequenced (Figure 2). It contains the ORF of 1344 bp coding for a protein of 447 amino acids with a calculated molecular weight of 50.7 kDa and pI value of 5.4. No intron was found. The opposite strand of the terminator region contains a potential ORF starting at position 2870. The possibly partial ORF codes for215 amino acid residues with a weak similarity to S.cerevisiae Bas1p and other transcription factors.

Complementation of the Saccharomyces cerevisiae sec19-1 temperature-sensitive mutation

To test the functionality of the cloned K. lactis and P. pastoris GDI1 genes and to perform a semi-quantitative comparison of functionalities of the cloned genes with the wild-type S. cerevisiae GDI1 gene, all three were cloned into YCpMET under the control of the MET25 promoter. The medium contained 1 mM methionine, which causes the cloned genes to be expressed on the weak basal level (Mumberg et al., 1994; Sangsoda et al., 1985). These plasmids and a vector control were introduced into the sec19-1 strain, and equal amounts and dilutions of cell suspensions were spotted onto selective plates at 24°C, 37°C and 38°C (Figure 3). Both the K. lactis and the P. pastoris GDI1 genes were able, and in a very similar way to the S. cerevisiae GDI1, to fully complement the temperature-sensitive phenotype of the sec19-1 strain NY1213. This is clearly attributable to the high homology between Gdi proteins found in various eukaryotic organisms (Figure 4).