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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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Mannosyl acceptor peptide preferences of Pmt4.In vitro O-mannosyltransferase activity was determined as described under ”Experimental Procedures.“ Mean ± S.D. values of three replicates are shown. A, peptide sequences of the mannosyl acceptors 418–440-bio, 401–420-bio, and Thr to Ala mutations thereof. B, mannosyltransferase activity of endogenous Pmt4 was measured using crude membranes from a pmt1 deletion mutant (pmt1/Pmt4). Standard reaction conditions were applied for the indicated acceptor peptides. Relative activities are displayed with respect to the peptide 401–420-bio for which activity was set to 100%. The average Dol-P-[3H]Man input was 33,314 dpm/reaction.
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Figure 5: Mannosyl acceptor peptide preferences of Pmt4.In vitro O-mannosyltransferase activity was determined as described under ”Experimental Procedures.“ Mean ± S.D. values of three replicates are shown. A, peptide sequences of the mannosyl acceptors 418–440-bio, 401–420-bio, and Thr to Ala mutations thereof. B, mannosyltransferase activity of endogenous Pmt4 was measured using crude membranes from a pmt1 deletion mutant (pmt1/Pmt4). Standard reaction conditions were applied for the indicated acceptor peptides. Relative activities are displayed with respect to the peptide 401–420-bio for which activity was set to 100%. The average Dol-P-[3H]Man input was 33,314 dpm/reaction.

Mentions: Our analysis revealed that in vitro properties of Pmt4 resemble those from human POMTs, but show some distinct features when compared with yeast Pmt1-Pmt2. To further explore similarities in acceptor preferences between POMTs and yeast Pmt4, in vitro O-mannosylation of peptide 401–420-bio-derived variants in which the four putative mannosylation sites were individually exchanged to alanine were analyzed (Fig. 5A). It was previously reported for the human POMT complex that changing Thr-404, Thr-406, and Thr-414 to Ala greatly reduced acceptor efficiency of peptide 401–420 (29). As shown in Fig. 5B, Pmt4-based transfer of [3H]mannose to peptides T404A, T406A, T414A, and T418A was significantly decreased with T414A showing the most pronounced effect (∼2% acceptor efficiency) (Fig. 5B).


Functional Similarities between the Protein O -Mannosyltransferases Pmt4 from Bakers' Yeast and Human POMT1 *
Mannosyl acceptor peptide preferences of Pmt4.In vitro O-mannosyltransferase activity was determined as described under ”Experimental Procedures.“ Mean ± S.D. values of three replicates are shown. A, peptide sequences of the mannosyl acceptors 418–440-bio, 401–420-bio, and Thr to Ala mutations thereof. B, mannosyltransferase activity of endogenous Pmt4 was measured using crude membranes from a pmt1 deletion mutant (pmt1/Pmt4). Standard reaction conditions were applied for the indicated acceptor peptides. Relative activities are displayed with respect to the peptide 401–420-bio for which activity was set to 100%. The average Dol-P-[3H]Man input was 33,314 dpm/reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016187&req=5

Figure 5: Mannosyl acceptor peptide preferences of Pmt4.In vitro O-mannosyltransferase activity was determined as described under ”Experimental Procedures.“ Mean ± S.D. values of three replicates are shown. A, peptide sequences of the mannosyl acceptors 418–440-bio, 401–420-bio, and Thr to Ala mutations thereof. B, mannosyltransferase activity of endogenous Pmt4 was measured using crude membranes from a pmt1 deletion mutant (pmt1/Pmt4). Standard reaction conditions were applied for the indicated acceptor peptides. Relative activities are displayed with respect to the peptide 401–420-bio for which activity was set to 100%. The average Dol-P-[3H]Man input was 33,314 dpm/reaction.
Mentions: Our analysis revealed that in vitro properties of Pmt4 resemble those from human POMTs, but show some distinct features when compared with yeast Pmt1-Pmt2. To further explore similarities in acceptor preferences between POMTs and yeast Pmt4, in vitro O-mannosylation of peptide 401–420-bio-derived variants in which the four putative mannosylation sites were individually exchanged to alanine were analyzed (Fig. 5A). It was previously reported for the human POMT complex that changing Thr-404, Thr-406, and Thr-414 to Ala greatly reduced acceptor efficiency of peptide 401–420 (29). As shown in Fig. 5B, Pmt4-based transfer of [3H]mannose to peptides T404A, T406A, T414A, and T418A was significantly decreased with T414A showing the most pronounced effect (∼2% acceptor efficiency) (Fig. 5B).

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