Genetic Analysis of Mutant Strains of Saccharomyces cerevisiae with Defects in Mannoprotein Synthesis

: Mannan defective ( mnn ) mutants have constituted a fundamental tool in the study of the structure and biosynthesis of mannoproteins in Saccharomyces cerevisiae . They were isolated by the group of Dr. C.E. Ballou by random mutagenesis, and a selection method using specific antibodies obtained against the wild-type strain. Initially, the mutants were characterized biochemically, and in subsequent years the genes in which they were mutated were identified. All of them encode membrane proteins that catalyze the transfer of mannoses to N-oligosaccharides, sometimes isolated or as part of complexes made up of several proteins. However, the specific mutation of each of these mutants has only been identified in the case of mnn3 . In this work, we have completed the characterization of the mutants by sequencing the mutated genes in each of them. As expected, they are point mutations that involve the change of one amino acid for another in the mutated protein, or for a stop signal, resulting in a truncated protein.


Introduction
The glycosylation process in eukaryotic cells has been extensively studied, and most steps have been almost completely deciphered. In these studies, the yeast Saccharomyces cerevisiae, a reference model organism in many laboratories, has played a central role. The process of glycosylation of N-oligosaccharides in S. cerevisiae is the same as that of higher eukaryotes in the early stages, which take place in the endoplasmic reticulum (ER) which lead to the synthesis of the so-called "inner core" [1]. However, in the stages that take place in the Golgi apparatus, the so-called "outer chain" is added. These stages are different from higher eukaryotes, because the outer chain contains exclusively mannose and lacks other monosaccharides. All N-linked oligosaccharides in S. cerevisiae are "high mannose" type. In the study of the glycosylation process in S. cerevisiae, the isolation and characterization of defective mutants in the process has been essential. Among them, two groups are worth highlighting: the alg mutants (Asparragine-linked glycosilation defective) affected in the stages of the ER [1], and mnn (mannan defective), affected in the stages that occur in the Golgi apparatus [2]. Figure 1 shows the defects of mnn mutants in Noligosacharides structure. Among the latter, mnn9 presents the most drastic defect because it blocks the addition of the whole outer chain in Golgi [2,3]. The rest of the mnn mutants have also been identified by different research groups working on glycosylation  studies [2,[4][5][6][7][8][9]. However, the genetic defect of mnn mutants has only been identified in the case of the mnn3 mutant [10]. The objective of this work is to complete the genetic characterization of the mnn mutants, identifying the mutations in each of them, by sequencing the mutated genes in the original strains and comparing the sequences with those of the parental strain.

Strains
The mutants mnn1, mnn2, mnn5, mnn6, mnn9, mnn10 used in this study were from the laboratory collection and were kindly supplied by Dr. C.E. Ballou some years ago. They were grown in liquid or solid YEPD medium, containing 1% yeast extract, 2% peptone, 2% glucose. For solid medium, 2% agar was added.

Extraction of Nucleic Acids
Yeast DNA minipreps were prepared as in [11]. Yeast cells were resuspended in 1 mL of solution I (EDTA 50 mM pH 7), centrifuged, and resuspended again in 1 mL of solution II (Tris-SO4 50 mM pH 9.3 + 1% mercaptoethanol). After 15 min at room temperature, they were centrifuged again and resuspended in 0.6 mL of solution III (sodium acetate 3 M). Then, 0.5 mL of equilibrate phenol was added and the mixture was incubated at room temperature for 30 min, with shaking. After centrifugation, the nucleic acids, recovered in the aqueous phase, were precipitated with 2 volumes of previously chilled isopropanol, washed with 70% ethanol, dried, and dissolved in Tris-EDTA buffer, pH 8.0.  MNN1: located at chromosome V (YER001W), 2289 bp. It encodes an integral membrane protein with alpha-1,3-mannosyltransferase activity. The mutation in mnn1 is a missense point mutation that changes G by A in DNA. It results in a change of cysteine by tyrosine in the protein (see Table 1). MNN2: located at chromosome II (YBR015C), 1794 bp. It encodes an integral membrane protein with alpha-1,2-mannosyltransferase activity over mannoses linked by alpha-1,6. The mutation in mnn2 is a missense point mutation that changes G by A in DNA. It results in a change of glycine by aspartic acid in the protein.

Identification of Mutations in MNN Genes Responsible for Synthesis of Outer Chain
MNN5: located at chromosome V(YJL186W), 1761 bp. It encodes an integral membrane protein with alpha-1,2-mannosyltransferase activity over some mannoses linked by alpha-1,2. The mutation in mnn5 is a nonsense point mutation that changes G by A in DNA. It results in a change of tryptophan by a stop signal protein, thus resulting in a truncated protein.
MNN6: located at chromosome XVI (YPL053C), 1341 bp. It encodes an integral membrane protein with mannosylphosphate transferase activity. The mutation in mnn6 is a frameshift mutation due to the insertion of AA. The change in the reading frame most probably results in a nonfunctional protein.
MNN9: located at chromosome XVI (YPL050C), 1188 bp. It encodes an integral membrane protein which is part of a complex involved in outer chain elongation. The mutation in mnn9 is a nonsense point mutation that changes (G by A) in DNA. It results in a change of tryptophan by a stop signal protein, thus resulting in a truncated protein.
MNN10: located at chromosome IV (YDR245W), 1182 bp. It encodes an integral membrane protein which is part of a complex involved in outer chain elongation. The mutation in mnn10 is a nonsense point mutation that changes G by A in DNA. It results in a change of tryptophan by a stop signal protein, thus resulting in a truncated protein.
As expected, most cases were point mutations with different effects: missense mutations, nonsense mutations, or frameshift mutations.
In the last few years, the mnn mutants have been widely characterized phenotypically. With this study, the genotypic characterization is completed.
Author Contributions: Conceptualization, funding acquisition, writing and editing, project administration, L.M.H.; methodology and resources, P.G.; investigation: P.G. and E.P.; supervision, A.M., R.V., and M.R. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.