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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.

No MeSH data available.


Related in: MedlinePlus

Dependence of the Pmt4 in vitro mannosyltransferase activity on the Dol-P-[3H]Man input.A and B, unless otherwise stated, in vitro mannosyltransferase activity was determined under standard conditions including 4 μg of the mannosyl acceptor GST-αDG as detailed under ”Experimental Procedures.“ A, crude membranes from strains SEY6210 (WT; squares, 0.5 μg/μl; diamonds, 0.25 μg/μl) and pmt4 expressing pRS423 (pmt4; triangles, 0.5 μg/μl) or pJK4-B1 (overexpression of Pmt4FLAG; circles, 0.5 μg/μl; see also Fig. 2B), were used as enzyme source and in vitro mannosyltransfer at the indicated donor substrate concentrations was determined. Results of independent experiments are shown in different shades. B, SEY6210 (WT) membranes were used and in vitro mannosyltransfer was determined at indicated time points for the standard reaction and for fresh Dol-P-[3H]Man addition every 15 min (indicated by arrows) (n = 2–5). The average Dol-P-[3H]Man input was between 18,000 and 32,000 dpm in the standard reaction and between 23,000 to 34,000 dpm for all consecutive additions.
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Figure 3: Dependence of the Pmt4 in vitro mannosyltransferase activity on the Dol-P-[3H]Man input.A and B, unless otherwise stated, in vitro mannosyltransferase activity was determined under standard conditions including 4 μg of the mannosyl acceptor GST-αDG as detailed under ”Experimental Procedures.“ A, crude membranes from strains SEY6210 (WT; squares, 0.5 μg/μl; diamonds, 0.25 μg/μl) and pmt4 expressing pRS423 (pmt4; triangles, 0.5 μg/μl) or pJK4-B1 (overexpression of Pmt4FLAG; circles, 0.5 μg/μl; see also Fig. 2B), were used as enzyme source and in vitro mannosyltransfer at the indicated donor substrate concentrations was determined. Results of independent experiments are shown in different shades. B, SEY6210 (WT) membranes were used and in vitro mannosyltransfer was determined at indicated time points for the standard reaction and for fresh Dol-P-[3H]Man addition every 15 min (indicated by arrows) (n = 2–5). The average Dol-P-[3H]Man input was between 18,000 and 32,000 dpm in the standard reaction and between 23,000 to 34,000 dpm for all consecutive additions.

Mentions: Increasing Dol-P-Man concentrations enhanced the in vitro transfer of [3H]mannose almost linearly (Fig. 3A). At all concentrations the amount of the [3H]mannose transferred to GST-αDG was proportional to the amount of the Pmt4 input (Fig. 3A, WT: 0.5 and 0.25 μg/μl of membranes) although relative incorporation levels did not increase to more than 25% (Figs. 2A and 3A). Likely explanations of this limited incorporation are the transfer of [3H]mannose to endogenous mannosyl acceptors by PMTs and other Dol-P-Man utilizing enzymes present in the crude membranes. This view is supported by the fact that overexpression of Pmt4FLAG steepened the level of [3H]mannose transferred to GST-αDG (Fig. 3A, Pmt4FLAG: 0.5 μg/μl of membranes), and that additions of fresh Dol-P-Man to the standard assay in 15-min intervals linearly increased incorporation of [3H]mannose into GST-αDG (Fig. 3B).


Functional Similarities between the Protein O -Mannosyltransferases Pmt4 from Bakers' Yeast and Human POMT1 *
Dependence of the Pmt4 in vitro mannosyltransferase activity on the Dol-P-[3H]Man input.A and B, unless otherwise stated, in vitro mannosyltransferase activity was determined under standard conditions including 4 μg of the mannosyl acceptor GST-αDG as detailed under ”Experimental Procedures.“ A, crude membranes from strains SEY6210 (WT; squares, 0.5 μg/μl; diamonds, 0.25 μg/μl) and pmt4 expressing pRS423 (pmt4; triangles, 0.5 μg/μl) or pJK4-B1 (overexpression of Pmt4FLAG; circles, 0.5 μg/μl; see also Fig. 2B), were used as enzyme source and in vitro mannosyltransfer at the indicated donor substrate concentrations was determined. Results of independent experiments are shown in different shades. B, SEY6210 (WT) membranes were used and in vitro mannosyltransfer was determined at indicated time points for the standard reaction and for fresh Dol-P-[3H]Man addition every 15 min (indicated by arrows) (n = 2–5). The average Dol-P-[3H]Man input was between 18,000 and 32,000 dpm in the standard reaction and between 23,000 to 34,000 dpm for all consecutive additions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Dependence of the Pmt4 in vitro mannosyltransferase activity on the Dol-P-[3H]Man input.A and B, unless otherwise stated, in vitro mannosyltransferase activity was determined under standard conditions including 4 μg of the mannosyl acceptor GST-αDG as detailed under ”Experimental Procedures.“ A, crude membranes from strains SEY6210 (WT; squares, 0.5 μg/μl; diamonds, 0.25 μg/μl) and pmt4 expressing pRS423 (pmt4; triangles, 0.5 μg/μl) or pJK4-B1 (overexpression of Pmt4FLAG; circles, 0.5 μg/μl; see also Fig. 2B), were used as enzyme source and in vitro mannosyltransfer at the indicated donor substrate concentrations was determined. Results of independent experiments are shown in different shades. B, SEY6210 (WT) membranes were used and in vitro mannosyltransfer was determined at indicated time points for the standard reaction and for fresh Dol-P-[3H]Man addition every 15 min (indicated by arrows) (n = 2–5). The average Dol-P-[3H]Man input was between 18,000 and 32,000 dpm in the standard reaction and between 23,000 to 34,000 dpm for all consecutive additions.
Mentions: Increasing Dol-P-Man concentrations enhanced the in vitro transfer of [3H]mannose almost linearly (Fig. 3A). At all concentrations the amount of the [3H]mannose transferred to GST-αDG was proportional to the amount of the Pmt4 input (Fig. 3A, WT: 0.5 and 0.25 μg/μl of membranes) although relative incorporation levels did not increase to more than 25% (Figs. 2A and 3A). Likely explanations of this limited incorporation are the transfer of [3H]mannose to endogenous mannosyl acceptors by PMTs and other Dol-P-Man utilizing enzymes present in the crude membranes. This view is supported by the fact that overexpression of Pmt4FLAG steepened the level of [3H]mannose transferred to GST-αDG (Fig. 3A, Pmt4FLAG: 0.5 μg/μl of membranes), and that additions of fresh Dol-P-Man to the standard assay in 15-min intervals linearly increased incorporation of [3H]mannose into GST-αDG (Fig. 3B).

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