The high osmolarity glycerol response (HOG) MAP kinase pathway controls localization of a yeast golgi glycosyltransferase

Abstract

The yeast alpha-1,3-mannosyltransferase (Mnn1p) is localized to the Golgi by independent transmembrane and lumenal domain signals. The lumenal domain is localized to the Golgi complex when expressed as a soluble form (Mnn1-s) by exchange of its transmembrane domain for a cleavable signal sequence (Graham, T. R., and V. A. Krasnov. 1995. Mol. Biol. Cell. 6:809-824). Mutants that failed to retain the lumenal domain in the Golgi complex, called lumenal domain retention (ldr) mutants, were isolated by screening mutagenized yeast colonies for those that secreted Mnn1-s. Two genes were identified by this screen, HOG1, a gene encoding a mitogen-activated protein kinase (MAPK) that functions in the high osmolarity glycerol (HOG) pathway, and LDR1. We have found that basal signaling through the HOG pathway is required to localize Mnn1-s to the Golgi in standard osmotic conditions. Mutations in HOG1 and LDR1 also perturb localization of intact Mnn1p, resulting in its loss from early Golgi compartments and a concomitant increase of Mnn1p in later Golgi compartments.

Figure 1

(a) Functional model for the compartmental organization of the yeast Golgi complex (Graham and Emr, 1991; Gaynor et al., 1994). (b) Schematic of the yeast α-1,3-mannosyltransferase (Mnn1p) and the fusion proteins used in this report. Wild-type Mnn1p, a type II integral membrane protein, is shown at the top. The lumenal domain is expressed in the secretory pathway as a soluble protein (Mnn1-s) by exchanging the transmembrane domain (TMD) and cytoplasmic tail (CT) for the cleavable signal peptide of CPY. The Mnn1p TMD and CT are fused to the secreted enzyme invertase and the resulting fusion protein (M39I) is used to examine TMD mediated localization; previous studies have indicated that the CT does not contribute to the localization of Mnn1p (Graham and Krasnov, 1995).

Figure 2

(a) Strains carrying null mutations in the PMR1 gene mislocalize and secrete Mnn1-s. Strains were streaked out and then analyzed by the colony blot assay as described in Materials and Methods. All of the strains except WT 2μ MNN1-s are carrying pMNN1-s. Strains marked with an * indicate that it is a strain with a genetic background other than SEY6210. The strain abbreviations in the figure are as follows: WT 2μ MNN1-s (SEY6210 pRS426 MNN1-s); WT (SEY6210); arf1Δ (6210 arf1Δ); chc1Δ* (GPY1103); pmr1Δ* (L3865); pmr1Δ (SEY6210 pmr1Δ); ret1-1 (TGY381); WT* (TGY383). (b) Summary of results from colony blot experiments with different secretory pathway mutants tested. All strains carry pMNN1-s. All vps strains are in the SEY6210 background. mnn2* and mnn5* are the LB1-16A and LB65-5D strains respectively. (c) The results of a complementation test between two ldr mutants (TRY121 and TRY320) is shown. All strains carry the pMNN1-s plasmid. (d) The percent Mnn1-s secreted from each strain was determined by Western blot as described in Materials and Methods. Mnn1-s is expressed at a slightly higher level than Mnn1p and has a different mobility by SDS-PAGE due to differences in N-glycosylation. This allowed us to distinguish Mnn1-s from endogenous Mnn1p in these experiments.

Figure 3

Strain TRY120 carries a mutant allele of HOG1. (a) Colony blot showing complementation of the TRY120 Ldr⁻ phenotype by pTR-26 and pHOG1. TRY120 pHOG1, TRY120 pTR-26, TRY120 pRS315, and WT (SEY6210 pRS315) were streaked out in duplicate and examined by colony blot. All strains carry pMNN1-s. (b) Primers that annealed to vector sequences on pTR-26 that flanked the yeast genomic insert were used to sequence the ends of the insert. These sequences were used in a BLAST search of the complete yeast genome database to reveal the genomic insert represented diagrammatically. The HOG1 gene is depicted as an open box and sites used to subclone it into the pRS313 vector to make pHOG1 are shown as well. (c) A diploid made from TRY120 crossed to AMY36 (hog1Δ) fails to grow on 1.0 M sorbitol plates. Wild-type strains SEY6210 and FY250 (WT*) were streaked onto a YPD plate containing 1.0 M sorbitol along with haploid strains JSY2092 (SEY6210 pbsΔ), AMY36 (hog1Δ*), and TRY120. Diploids were made by crossing TRY120 with JSY2092 (TRY120/JSY2092) and AMY36 (TRY120/ AMY36) and these diploids were also streaked on the 1.0 M sorbitol to test for complementation of the pbs2Δ- and hog1Δ-associated growth defects, respectively.

Figure 8

Model for the potential role of the HOG and PKC-MPK pathways in regulating cell wall biosynthesis. The HOG pathway is required for Golgi localization of Mnn1-s, controls the distribution of Mnn1p between early and late Golgi compartments, and upregulates the activity of an exoglucanase, possibly in response to excess rigidity in the cell wall. Conversely, the PKC-MPK pathway is thought to sense a weakened cell wall and respond by upregulating the transcription of cell wall biosynthetic enzymes. See Discussion for additional details.

Figure 4

The HOG1 gene does not complement the Ldr⁻ phenotype in TRY220. (a) WT (SEY6210 pMNN1-s), TRY220 pMNN1-s, and TRY220 pMNN1-s pTR-26 were streaked out in duplicate and tested by colony blot. (b) Summary table of results from testing TRY120 (hog1-11) and TRY220 (with or without) a plasmid borne copy of the HOG1 gene for growth on 1 M sorbitol plates and for Mnn1-s secretion by colony blot. Other ldr mutants were tested for growth on 1 M sorbitol. s indicates Mnn1-s was secreted and ns indicates Mnn1-s was not secreted.

Figure 5

Mutations in the HOG1 gene do not perturb the transmembrane domain-mediated localization mechanism. Wild-type, hog1Δ, pbs2Δ, pmr1Δ, hog1-11, and ldr1-2 cells expressing M39I on a single-copy plasmid (pCM39S) were subjected to a liquid invertase assay (Bankaitis et al., 1986) to quantitate the level of M39I mislocalized to the cell surface.

Figure 6

Sucrose gradient fractionation profiles of Golgi membranes from wild-type, hog1-11, and ldr1-2 cells. Differential centrifugation, sucrose gradient fractionation, and enzyme assays were performed as described in Materials and Methods. Fractionation profiles of Kex2p, GDPase, and Mnn1p from (a) WT (SEY6210), (b) TRY120 (hog1-11), and (c) TRY220 (ldr1-2) cells were generated from the average of three experiments performed for each strain. The amount of each marker in a particular fraction was plotted as percentage of the total found in all the fractions. Quantitation of Western blots was done as described in Materials and Methods.

Figure 7

Examination of other mutants in the HOG pathway for the Ldr⁻ phenotype. Strains harboring null mutations in PBS2 (a) SSK2 SSK22 SHO1 (b) SSK1 (c) and SHO1 (d) were tested for Mnn1-s secretion by the colony blot assay. A hog1 mutant and isogenic wild-type strains were included in each plate. Strains marked with an asterisk (*) are in the W303 background and all other strains are in the SEY6210 background. All strains assayed are carrying pMNN1-s.