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

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.

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Detergent requirements of Pmt4 and Pmt1–2 complexes.In vitro O-mannosyltransferase activity was determined as detailed under ”Experimental Procedures.“ Mean ± S.D. values of three replicates are shown. A, amino acid sequence of peptide 401–420-bio. B and C, dependence of mannosyltransferase activity on detergent concentrations for β-OTG (B) or Triton X-100 (C) is shown for Pmt4 (enzyme source: membranes from a pmt1 deletion strain; pmt1/Pmt4) and for Pmt1-Pmt2 complexes (enzyme source: membranes from a pmt4 deletion strain; pmt4/Pmt1/2) using the 401–420-bio peptide. Detergent concentrations are indicated as a function of the critical micelle concentration (cmc). The average Dol-P-[3H]Man input was 23,357 (B) and 33,225 dpm (C) per reaction.
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Figure 4: Detergent requirements of Pmt4 and Pmt1–2 complexes.In vitro O-mannosyltransferase activity was determined as detailed under ”Experimental Procedures.“ Mean ± S.D. values of three replicates are shown. A, amino acid sequence of peptide 401–420-bio. B and C, dependence of mannosyltransferase activity on detergent concentrations for β-OTG (B) or Triton X-100 (C) is shown for Pmt4 (enzyme source: membranes from a pmt1 deletion strain; pmt1/Pmt4) and for Pmt1-Pmt2 complexes (enzyme source: membranes from a pmt4 deletion strain; pmt4/Pmt1/2) using the 401–420-bio peptide. Detergent concentrations are indicated as a function of the critical micelle concentration (cmc). The average Dol-P-[3H]Man input was 23,357 (B) and 33,225 dpm (C) per reaction.

Mentions: Our analysis revealed that in the presence of β-OTG as a detergent yeast Pmt4 can mannosylate the mammalian POMT substrate GST-αDG (Fig. 2A). In a pmt4Δ mutant, other O-mannosyltransferase activities were below the detection limits of the assay (Fig. 2A), although the Pmt1-Pmt2 complex is fully active in vitro in the absence of Pmt4 (with Triton X-100 as detergent) (8). In contrast, our previous studies showed in vitro mannosylation of the acceptor peptide bio-YATAV by Pmt1-Pmt2, but not by Pmt4, in the presence of the detergent Triton X-100 (17, 32). To further address in vitro mannosyl acceptor specificities of the yeast PMT family members, we first analyzed the detergent requirements in more detail. To individually record endogenous Pmt4 and Pmt1-Pmt2 activities, membranes from pmt1 and pmt4 deletion mutants, respectively, were used as an enzyme source. For vivid depiction, membranes from these strains are identified as pmt1/Pmt4 and pmt4/Pmt1/2 in Table 1 and Fig. 4. Under the conditions applied, in mutant pmt1Δ in vitro enzymatic activity of Pmt2 and other PMT1 and PMT2 family members is negligibly small (17). Even in the presence of Triton X-100, GST-αDG did not serve as acceptor substrate of Pmt1-Pmt2 (data not shown). But, a proven POMT in vitro substrate, the αDG-derived synthetic peptide including amino acids 401 to 420 (29) (Fig. 4A) qualified as a more general mannosyl acceptor. Following the transfer of [3H]mannose from Dol-P-Man to peptide 401–420-bio showed that Pmt4 and Pmt1-Pmt2 complexes were both active with β-OTG as detergent, and showed similar β-OTG optima at ∼1.5 × critical micelle concentration (∼0.42%). Although protein levels of Pmt4 are at least 2–3 times lower when compared with Pmt1 and Pmt2 (data not shown), Pmt4 activity was significantly higher (Fig. 4B). In contrast, Pmt4 was almost inactive with Triton X-100 as detergent, whereas Pmt1-Pmt2 activity was characterized by an optimal curve with the highest [3H]mannose transfer at ∼0.12% Triton X-100 (∼10 × critical micelle concentration; Fig. 4C).


Functional Similarities between the Protein O -Mannosyltransferases Pmt4 from Bakers' Yeast and Human POMT1 *
Detergent requirements of Pmt4 and Pmt1–2 complexes.In vitro O-mannosyltransferase activity was determined as detailed under ”Experimental Procedures.“ Mean ± S.D. values of three replicates are shown. A, amino acid sequence of peptide 401–420-bio. B and C, dependence of mannosyltransferase activity on detergent concentrations for β-OTG (B) or Triton X-100 (C) is shown for Pmt4 (enzyme source: membranes from a pmt1 deletion strain; pmt1/Pmt4) and for Pmt1-Pmt2 complexes (enzyme source: membranes from a pmt4 deletion strain; pmt4/Pmt1/2) using the 401–420-bio peptide. Detergent concentrations are indicated as a function of the critical micelle concentration (cmc). The average Dol-P-[3H]Man input was 23,357 (B) and 33,225 dpm (C) per reaction.
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Related In: Results  -  Collection

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Figure 4: Detergent requirements of Pmt4 and Pmt1–2 complexes.In vitro O-mannosyltransferase activity was determined as detailed under ”Experimental Procedures.“ Mean ± S.D. values of three replicates are shown. A, amino acid sequence of peptide 401–420-bio. B and C, dependence of mannosyltransferase activity on detergent concentrations for β-OTG (B) or Triton X-100 (C) is shown for Pmt4 (enzyme source: membranes from a pmt1 deletion strain; pmt1/Pmt4) and for Pmt1-Pmt2 complexes (enzyme source: membranes from a pmt4 deletion strain; pmt4/Pmt1/2) using the 401–420-bio peptide. Detergent concentrations are indicated as a function of the critical micelle concentration (cmc). The average Dol-P-[3H]Man input was 23,357 (B) and 33,225 dpm (C) per reaction.
Mentions: Our analysis revealed that in the presence of β-OTG as a detergent yeast Pmt4 can mannosylate the mammalian POMT substrate GST-αDG (Fig. 2A). In a pmt4Δ mutant, other O-mannosyltransferase activities were below the detection limits of the assay (Fig. 2A), although the Pmt1-Pmt2 complex is fully active in vitro in the absence of Pmt4 (with Triton X-100 as detergent) (8). In contrast, our previous studies showed in vitro mannosylation of the acceptor peptide bio-YATAV by Pmt1-Pmt2, but not by Pmt4, in the presence of the detergent Triton X-100 (17, 32). To further address in vitro mannosyl acceptor specificities of the yeast PMT family members, we first analyzed the detergent requirements in more detail. To individually record endogenous Pmt4 and Pmt1-Pmt2 activities, membranes from pmt1 and pmt4 deletion mutants, respectively, were used as an enzyme source. For vivid depiction, membranes from these strains are identified as pmt1/Pmt4 and pmt4/Pmt1/2 in Table 1 and Fig. 4. Under the conditions applied, in mutant pmt1Δ in vitro enzymatic activity of Pmt2 and other PMT1 and PMT2 family members is negligibly small (17). Even in the presence of Triton X-100, GST-αDG did not serve as acceptor substrate of Pmt1-Pmt2 (data not shown). But, a proven POMT in vitro substrate, the αDG-derived synthetic peptide including amino acids 401 to 420 (29) (Fig. 4A) qualified as a more general mannosyl acceptor. Following the transfer of [3H]mannose from Dol-P-Man to peptide 401–420-bio showed that Pmt4 and Pmt1-Pmt2 complexes were both active with β-OTG as detergent, and showed similar β-OTG optima at ∼1.5 × critical micelle concentration (∼0.42%). Although protein levels of Pmt4 are at least 2–3 times lower when compared with Pmt1 and Pmt2 (data not shown), Pmt4 activity was significantly higher (Fig. 4B). In contrast, Pmt4 was almost inactive with Triton X-100 as detergent, whereas Pmt1-Pmt2 activity was characterized by an optimal curve with the highest [3H]mannose transfer at ∼0.12% Triton X-100 (∼10 × critical micelle concentration; Fig. 4C).

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