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Functional Similarities between the Protein O -Mannosyltransferases Pmt4 from Bakers' Yeast and Human POMT1 *

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ABSTRACT

Protein O-mannosylation is an essential post-translational modification. It is initiated in the endoplasmic reticulum by a family of protein O-mannosyltransferases that are conserved from yeast (PMTs) to human (POMTs). The degree of functional conservation between yeast and human protein O-mannosyltransferases is uncharacterized. In bakers' yeast, the main in vivo activities are due to heteromeric Pmt1-Pmt2 and homomeric Pmt4 complexes. Here we describe an enzymatic assay that allowed us to monitor Pmt4 activity in vitro. We demonstrate that detergent requirements and acceptor substrates of yeast Pmt4 are different from Pmt1-Pmt2, but resemble that of human POMTs. Furthermore, we mimicked two POMT1 amino acid exchanges (G76R and V428D) that result in severe congenital muscular dystrophies in humans, in yeast Pmt4 (I112R and I435D). In vivo and in vitro analyses showed that general features such as protein stability of the Pmt4 variants were not significantly affected, however, the mutants proved largely enzymatically inactive. Our results demonstrate functional and biochemical similarities between POMT1 and its orthologue from bakers' yeast Pmt4.

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Pmt4 in vitro mannosyltransferase activity.A, crude membranes from wild-type strain SEY6210 (WT) and pmt4  mutant transformed with pRS423 (vector) or pJK4-B1 (Pmt4FLAG) were tested for in vitro mannosyltransferase activity. The standard assay containing 29,067 dpm of Dol-P-[3H]Man per reaction was performed as described under ”Experimental Procedures.“ Mean ± S.D. values of at least three independent experiments are shown. B, quantification of the Pmt4 content of the membranes used in A. Western blottings were probed with anti-Pmt4 and anti-Sec61 antibodies. Signal intensities were quantified as outlined under ”Experimental Procedures.“ The indicated numbers represent the relative Pmt4 content (ratio Pmt4:Sec61) with reference to WT, which was set as 1. C–H, characterization of the Pmt4 in vitro mannosyltransferase activity. Standard assays containing between 20,000 and 30,000 dpm of Dol-P-[3H]Man per reaction were performed. Mean ± S.D. values of independent experiments are shown (n = 2 to 4). Dependence of the in vitro mannosyltransfer reaction on the concentration of the mannose acceptor GST-αDG (C), the reaction time (D), the membrane protein input (E), the reaction temperature (F), the pH (G), and the presence of divalent cations and EDTA at the depicted concentrations (H) was determined using SEY6210 membranes as an enzyme source.
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Figure 2: Pmt4 in vitro mannosyltransferase activity.A, crude membranes from wild-type strain SEY6210 (WT) and pmt4 mutant transformed with pRS423 (vector) or pJK4-B1 (Pmt4FLAG) were tested for in vitro mannosyltransferase activity. The standard assay containing 29,067 dpm of Dol-P-[3H]Man per reaction was performed as described under ”Experimental Procedures.“ Mean ± S.D. values of at least three independent experiments are shown. B, quantification of the Pmt4 content of the membranes used in A. Western blottings were probed with anti-Pmt4 and anti-Sec61 antibodies. Signal intensities were quantified as outlined under ”Experimental Procedures.“ The indicated numbers represent the relative Pmt4 content (ratio Pmt4:Sec61) with reference to WT, which was set as 1. C–H, characterization of the Pmt4 in vitro mannosyltransferase activity. Standard assays containing between 20,000 and 30,000 dpm of Dol-P-[3H]Man per reaction were performed. Mean ± S.D. values of independent experiments are shown (n = 2 to 4). Dependence of the in vitro mannosyltransfer reaction on the concentration of the mannose acceptor GST-αDG (C), the reaction time (D), the membrane protein input (E), the reaction temperature (F), the pH (G), and the presence of divalent cations and EDTA at the depicted concentrations (H) was determined using SEY6210 membranes as an enzyme source.

Mentions: The PMT4 family mannosyltransferases from bakers' yeast (Pmt4) and human (POMT1) show a high degree of conservation (Fig. 1) (26). To establish an in vitro assay to monitor Pmt4-mediated mannosyl transfer, we thus tested conditions previously used for in vitro activity measurements of the mammalian POMTs (31). Indeed, the use of GST-tagged α-dystroglycan mucin domain (GST-αDG) as acceptor substrate and β-octylthioglucoside (β-OTG) as detergent enabled detection of yeast Pmt4 activity in vitro (Fig. 2A) (19). In a reaction mixture containing [3H]mannose-labeled Dol-P-Man as donor substrate and crude membranes isolated from wild-type yeast strain SEY6210 as enzyme source, typically 10 to 15% of the tritiated mannose were transferred to GST-αDG, but not to GST alone (Fig. 2A). Remarkably, this assay exclusively monitored Pmt4 activity because in total membranes from a pmt4Δ mutant strain mannosyltransferase activity was around background level, although all PMT1 and PMT2 family members were present (Fig. 2A). Expression of a FLAG-tagged version of Pmt4 (Pmt4FLAG) from a multicopy plasmid restored in vitro mannosyltransferase activity in the latter strain. The roughly 2-fold increase in mannosyl transfer (Fig. 2A) correlated well with 2.1-fold higher enzyme content when compared with wild-type membrane preparations (Fig. 2B).


Functional Similarities between the Protein O -Mannosyltransferases Pmt4 from Bakers' Yeast and Human POMT1 *
Pmt4 in vitro mannosyltransferase activity.A, crude membranes from wild-type strain SEY6210 (WT) and pmt4  mutant transformed with pRS423 (vector) or pJK4-B1 (Pmt4FLAG) were tested for in vitro mannosyltransferase activity. The standard assay containing 29,067 dpm of Dol-P-[3H]Man per reaction was performed as described under ”Experimental Procedures.“ Mean ± S.D. values of at least three independent experiments are shown. B, quantification of the Pmt4 content of the membranes used in A. Western blottings were probed with anti-Pmt4 and anti-Sec61 antibodies. Signal intensities were quantified as outlined under ”Experimental Procedures.“ The indicated numbers represent the relative Pmt4 content (ratio Pmt4:Sec61) with reference to WT, which was set as 1. C–H, characterization of the Pmt4 in vitro mannosyltransferase activity. Standard assays containing between 20,000 and 30,000 dpm of Dol-P-[3H]Man per reaction were performed. Mean ± S.D. values of independent experiments are shown (n = 2 to 4). Dependence of the in vitro mannosyltransfer reaction on the concentration of the mannose acceptor GST-αDG (C), the reaction time (D), the membrane protein input (E), the reaction temperature (F), the pH (G), and the presence of divalent cations and EDTA at the depicted concentrations (H) was determined using SEY6210 membranes as an enzyme source.
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Figure 2: Pmt4 in vitro mannosyltransferase activity.A, crude membranes from wild-type strain SEY6210 (WT) and pmt4 mutant transformed with pRS423 (vector) or pJK4-B1 (Pmt4FLAG) were tested for in vitro mannosyltransferase activity. The standard assay containing 29,067 dpm of Dol-P-[3H]Man per reaction was performed as described under ”Experimental Procedures.“ Mean ± S.D. values of at least three independent experiments are shown. B, quantification of the Pmt4 content of the membranes used in A. Western blottings were probed with anti-Pmt4 and anti-Sec61 antibodies. Signal intensities were quantified as outlined under ”Experimental Procedures.“ The indicated numbers represent the relative Pmt4 content (ratio Pmt4:Sec61) with reference to WT, which was set as 1. C–H, characterization of the Pmt4 in vitro mannosyltransferase activity. Standard assays containing between 20,000 and 30,000 dpm of Dol-P-[3H]Man per reaction were performed. Mean ± S.D. values of independent experiments are shown (n = 2 to 4). Dependence of the in vitro mannosyltransfer reaction on the concentration of the mannose acceptor GST-αDG (C), the reaction time (D), the membrane protein input (E), the reaction temperature (F), the pH (G), and the presence of divalent cations and EDTA at the depicted concentrations (H) was determined using SEY6210 membranes as an enzyme source.
Mentions: The PMT4 family mannosyltransferases from bakers' yeast (Pmt4) and human (POMT1) show a high degree of conservation (Fig. 1) (26). To establish an in vitro assay to monitor Pmt4-mediated mannosyl transfer, we thus tested conditions previously used for in vitro activity measurements of the mammalian POMTs (31). Indeed, the use of GST-tagged α-dystroglycan mucin domain (GST-αDG) as acceptor substrate and β-octylthioglucoside (β-OTG) as detergent enabled detection of yeast Pmt4 activity in vitro (Fig. 2A) (19). In a reaction mixture containing [3H]mannose-labeled Dol-P-Man as donor substrate and crude membranes isolated from wild-type yeast strain SEY6210 as enzyme source, typically 10 to 15% of the tritiated mannose were transferred to GST-αDG, but not to GST alone (Fig. 2A). Remarkably, this assay exclusively monitored Pmt4 activity because in total membranes from a pmt4Δ mutant strain mannosyltransferase activity was around background level, although all PMT1 and PMT2 family members were present (Fig. 2A). Expression of a FLAG-tagged version of Pmt4 (Pmt4FLAG) from a multicopy plasmid restored in vitro mannosyltransferase activity in the latter strain. The roughly 2-fold increase in mannosyl transfer (Fig. 2A) correlated well with 2.1-fold higher enzyme content when compared with wild-type membrane preparations (Fig. 2B).

View Article: PubMed Central - PubMed

ABSTRACT

Protein O-mannosylation is an essential post-translational modification. It is initiated in the endoplasmic reticulum by a family of protein O-mannosyltransferases that are conserved from yeast (PMTs) to human (POMTs). The degree of functional conservation between yeast and human protein O-mannosyltransferases is uncharacterized. In bakers' yeast, the main in vivo activities are due to heteromeric Pmt1-Pmt2 and homomeric Pmt4 complexes. Here we describe an enzymatic assay that allowed us to monitor Pmt4 activity in vitro. We demonstrate that detergent requirements and acceptor substrates of yeast Pmt4 are different from Pmt1-Pmt2, but resemble that of human POMTs. Furthermore, we mimicked two POMT1 amino acid exchanges (G76R and V428D) that result in severe congenital muscular dystrophies in humans, in yeast Pmt4 (I112R and I435D). In vivo and in vitro analyses showed that general features such as protein stability of the Pmt4 variants were not significantly affected, however, the mutants proved largely enzymatically inactive. Our results demonstrate functional and biochemical similarities between POMT1 and its orthologue from bakers' yeast Pmt4.

No MeSH data available.


Related in: MedlinePlus