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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.


In vitro mannosyltransferase activity of Pmt4 mutant proteins.A, in vitro activities of Pmt4 and the variants thereof. In vitro mannosyltransferase activity was determined as specified under ”Experimental Procedures.“ Mean ± S.D. values of at least three independent experiments are shown as relative activities referring to Pmt4FLAG for which activity was set to 100%. Crude membranes from pmt4  mutants expressing pJK4-B1 (Pmt4), pMS1 (I112R), pMS2 (I435D), or pDB6 (I435V) were used as enzyme source. The average Dol-P-[3H]Man input was 33,942 dpm/reaction for Pmt4, I112R, and I435D or 29,758 dpm/reaction for I435V. Maximal activities of about 7,000 dpm could be measured for wild-type Pmt4 with background values of around 100 dpm when no acceptor was added. Thus, activities below 1.5% of WT could not be evaluated. B, Western blotting analysis based quantification of Pmt4 variants in the membrane preparations which were used in A. Blots were probed with anti-Pmt4 and anti-Sec61 antibodies. Signal intensities were detected and analyzed as outlined under ”Experimental Procedures.“ The indicated numbers represent the relative Pmt4 content (ratio Pmt4:Sec61) with reference to wild-type Pmt4, which was set as 1.
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Figure 6: In vitro mannosyltransferase activity of Pmt4 mutant proteins.A, in vitro activities of Pmt4 and the variants thereof. In vitro mannosyltransferase activity was determined as specified under ”Experimental Procedures.“ Mean ± S.D. values of at least three independent experiments are shown as relative activities referring to Pmt4FLAG for which activity was set to 100%. Crude membranes from pmt4 mutants expressing pJK4-B1 (Pmt4), pMS1 (I112R), pMS2 (I435D), or pDB6 (I435V) were used as enzyme source. The average Dol-P-[3H]Man input was 33,942 dpm/reaction for Pmt4, I112R, and I435D or 29,758 dpm/reaction for I435V. Maximal activities of about 7,000 dpm could be measured for wild-type Pmt4 with background values of around 100 dpm when no acceptor was added. Thus, activities below 1.5% of WT could not be evaluated. B, Western blotting analysis based quantification of Pmt4 variants in the membrane preparations which were used in A. Blots were probed with anti-Pmt4 and anti-Sec61 antibodies. Signal intensities were detected and analyzed as outlined under ”Experimental Procedures.“ The indicated numbers represent the relative Pmt4 content (ratio Pmt4:Sec61) with reference to wild-type Pmt4, which was set as 1.

Mentions: Under standard conditions with GST-αDG as mannosyl acceptor substrate, mannosyltransferase activity of the different enzyme variants was analyzed. Crude membranes were hence prepared from a pmt4 mutant expressing FLAG-tagged versions of either wild-type or mutant Pmt4. Western blotting analysis revealed that the steady-state levels of Pmt4FLAG and mutants I112R, I435D, and I435V did not significantly differ from each other (Fig. 6B). In these preparations, mannosyltransferase activity of mutant I112R was highly reduced (by ∼97.5% when compared with the quantifiable activity of the wild-type enzyme). Enzymatic activity of mutant I435D could not be detected within the limits of the assay, whereas mutant I435V was at wild-type level (Fig. 6A).


Functional Similarities between the Protein O -Mannosyltransferases Pmt4 from Bakers' Yeast and Human POMT1 *
In vitro mannosyltransferase activity of Pmt4 mutant proteins.A, in vitro activities of Pmt4 and the variants thereof. In vitro mannosyltransferase activity was determined as specified under ”Experimental Procedures.“ Mean ± S.D. values of at least three independent experiments are shown as relative activities referring to Pmt4FLAG for which activity was set to 100%. Crude membranes from pmt4  mutants expressing pJK4-B1 (Pmt4), pMS1 (I112R), pMS2 (I435D), or pDB6 (I435V) were used as enzyme source. The average Dol-P-[3H]Man input was 33,942 dpm/reaction for Pmt4, I112R, and I435D or 29,758 dpm/reaction for I435V. Maximal activities of about 7,000 dpm could be measured for wild-type Pmt4 with background values of around 100 dpm when no acceptor was added. Thus, activities below 1.5% of WT could not be evaluated. B, Western blotting analysis based quantification of Pmt4 variants in the membrane preparations which were used in A. Blots were probed with anti-Pmt4 and anti-Sec61 antibodies. Signal intensities were detected and analyzed as outlined under ”Experimental Procedures.“ The indicated numbers represent the relative Pmt4 content (ratio Pmt4:Sec61) with reference to wild-type Pmt4, which was set as 1.
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Figure 6: In vitro mannosyltransferase activity of Pmt4 mutant proteins.A, in vitro activities of Pmt4 and the variants thereof. In vitro mannosyltransferase activity was determined as specified under ”Experimental Procedures.“ Mean ± S.D. values of at least three independent experiments are shown as relative activities referring to Pmt4FLAG for which activity was set to 100%. Crude membranes from pmt4 mutants expressing pJK4-B1 (Pmt4), pMS1 (I112R), pMS2 (I435D), or pDB6 (I435V) were used as enzyme source. The average Dol-P-[3H]Man input was 33,942 dpm/reaction for Pmt4, I112R, and I435D or 29,758 dpm/reaction for I435V. Maximal activities of about 7,000 dpm could be measured for wild-type Pmt4 with background values of around 100 dpm when no acceptor was added. Thus, activities below 1.5% of WT could not be evaluated. B, Western blotting analysis based quantification of Pmt4 variants in the membrane preparations which were used in A. Blots were probed with anti-Pmt4 and anti-Sec61 antibodies. Signal intensities were detected and analyzed as outlined under ”Experimental Procedures.“ The indicated numbers represent the relative Pmt4 content (ratio Pmt4:Sec61) with reference to wild-type Pmt4, which was set as 1.
Mentions: Under standard conditions with GST-αDG as mannosyl acceptor substrate, mannosyltransferase activity of the different enzyme variants was analyzed. Crude membranes were hence prepared from a pmt4 mutant expressing FLAG-tagged versions of either wild-type or mutant Pmt4. Western blotting analysis revealed that the steady-state levels of Pmt4FLAG and mutants I112R, I435D, and I435V did not significantly differ from each other (Fig. 6B). In these preparations, mannosyltransferase activity of mutant I112R was highly reduced (by ∼97.5% when compared with the quantifiable activity of the wild-type enzyme). Enzymatic activity of mutant I435D could not be detected within the limits of the assay, whereas mutant I435V was at wild-type level (Fig. 6A).

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.