Interaction between the C termini of Alg13 and Alg14 mediates formation of the active UDP-N-acetylglucosamine transferase complex

Abstract

The second step of eukaryotic N-linked glycosylation in endoplasmic reticulum is catalyzed by an UDP-N-acetylglucosamine transferase that is comprised of two subunits, Alg13 and Alg14. The interaction between Alg13 and 14 is crucial for UDP-GlcNAc transferase activity, so formation of the Alg13/14 complex is likely to play a key role in the regulation of N-glycosylation. Using a combination of bioinformatics and molecular biological methods, we have undertaken a functional analysis of yeast Alg13 and Alg14 proteins to elucidate the mechanism of their interaction. Our mutational studies demonstrated that a short C-terminal alpha-helix of Alg13 is required for interaction with Alg14 and for enzyme activity. Electrostatic surface views of the modeled Alg13/14 complex suggest the presence of a hydrophobic cleft in Alg14 that provides a pocket for the Alg13 C-terminal alpha-helix. Co-immunoprecipitation assays confirmed the C-terminal three amino acids of Alg14 are required for maintaining the integrity of Alg13/Alg14 complex, and this depends on their hydrophobicity. Modeling studies place these three Alg14 residues at the entrance of the hydrophobic-binding pocket, suggesting their role in the stabilization of the interaction between the C termini of Alg13 and Alg14. Together, these results demonstrate that formation of this hetero-oligomeric complex is mediated by a short C-terminal alpha-helix of Alg13 in cooperation with the last three amino acids of Alg14. In addition, deletion of the N-terminal beta-strand of Alg13 caused the destruction of protein, indicating the structural importance of this region in protein stability.

FIGURE 1.

Theoretical structure of the Alg13/14 complex is structurally similar to MurG. A and B, stereo structures of yeast Alg13/14 complex and E. coli MurG (1F0K). The ribbon representation shows the predicted yeast Alg13/14 complex adopting the GT-B fold with a structure very similar to that of MurG (see “Experimental Procedures” for details of how these models were generated). For both structures, the β-strands in the two Rossmann fold subdomains are drawn in yellow, and the flanking α-helices in red. The linker peptide that connects the two Rossmann fold domains in MurG but that is absent from Alg13/14 complex is drawn in green. The extended C-terminal α-helix of MurG or Alg13 is drawn in blue. Termini of all proteins are indicated by arrows. C and D, sequence alignment of Alg13 and 14 against MurG and their topological diagrams in modeled Alg13/14 complex. The Swiss-Prot accession number is given in parentheses. C, yeast Alg14 (P38242) and the N-terminal region of E. coli MurG (P17443) is aligned and manually mapped as described (8). The secondary structure of the MurG (25) is shown above the alignment. The secondary structure predictions used for Alg14 (8) are presented in schematic form under the alignment. The predicted transmembrane at the N-terminal of Alg14 is shown as a white box. D, an alignment between yeast Alg13 (P53178) and the C-terminal region of E. coli MurG (P17443) is mapped as described (8). The secondary structure of the MurG (25) is shown above the alignment. The experimentally determined secondary structure used for Alg13 (2jzc) (15) is presented in a scheme under the alignment.

FIGURE 2.

The C-terminal α-helix of yeast Alg13 is required for viability and for interaction with Alg14. A, C-terminal sequence of yeast Alg13 protein. The predicted α-helix is boxed. B, yeast strain (XGY154) with ALG13 under control of the glucose-repressible GAL1 promoter (P_GAL1) was transformed with plasmids containing FLAG-ALG13 (pXG211), FLAG-alg13-CΔ3 (pSA4), or FLAG-alg13-CΔ15 (pSA6), and streaked onto YPA plates supplemented with galactose (right panel) or glucose (left panel). Cells were incubated for 2 days at 30 °C. C, whole cell detergent extracts were prepared from a wild-type strain (W303a) that co-expresses HA-ALG14 (pXG202) and FLAG-alg13-CΔ15 (pSA6) or FLAG-ALG13 (pXG211). Samples were immunoprecipitated with anti-HA affinity matrix (lanes 1–3) or quantitatively analyzed for the presence of Alg13 and Alg13-CΔ15 proteins by Western blotting (lanes 4–6). Proteins or extracts were separated by 12% SDS-PAGE, immunoblotted with rabbit anti-FLAG antibodies, and detected by chemiluminescence as described under “Experimental Procedures.”

FIGURE 3.

The C-terminal amino acids of Alg14 are required for viability and interaction with Alg13. A, C-terminal sequence of yeast Alg14 protein. The predicted β-strand region is boxed. B, yeast strain (XGY151) with ALG14 under control of the glucose-repressible GAL1 promoter (P_GAL1) was transformed with plasmids containing HA-ALG14 (pXG202), HA-alg14-CΔ3 (pSA7), or HA-alg14-CΔ12 (pSA8), and streaked onto YPA plates supplemented with galactose (right panel) or glucose (left panel). Cells were incubated for 2 days at 30 °C. C, a strain that contains a C-terminal triple FLAG-tagged ALG13 at the chromosomal ALG13 locus (XGY155) was transformed with plasmids encoding HA-ALG14 (pXG202) or HA-alg14-CΔ3 (pSA7) or HA-alg14-CΔ12 (pSA8). ER-enriched membrane fractions (P20) from the transformed cells were prepared as described under “Experimental Procedures.” Protein (80 μg) from each sample was separated by 12% SDS-PAGE and quantitatively analyzed for HA-tagged Alg14 proteins by immunoblotting with a mouse anti-HA monoclonal antibody conjugated to peroxidase. D, whole cell detergent extracts were prepared from same strains used in the Western blotting assay described above. Samples were immunoprecipitated with anti-HA affinity matrix (lanes 1–4) or directly analyzed for the presence of triple FLAG-tagged Alg13p by Western blot (lanes 5–8). Immunoprecipitated proteins or extracts were separated by 12% SDS-PAGE, immunoblotted with rabbit anti-FLAG antibodies, and detected by chemiluminescence as described under “Experimental Procedures.”

FIGURE 4.

Molecular surface view of Alg14 proposes a hydrophobic cleft that serves as binding pocket for the C-terminal α-helix of Alg13. Molecular surface of yeast Alg13 and 14 proteins colored according to the residue hydrophobicity in which the high to low lipophilicity scale corresponds to the color ramp from blue to brown. Upper panels, top (A) and side (B) views of Alg14. The patch circled in white indicates the putative binding pocket for the C-terminal α-helix of Alg13. The arrow in red indicates the last three hydrophobic residues of Alg14. The structure of the C-terminal region of Alg13, including the last α-helix, is shown in the scheme; Lower panels, top (C) and side (D) views of the C-terminal region of Alg13, including the proposed α-helix that interacts with Alg14 at the binding pocket. The window represented in (C) panel indicates the back view of the C-terminal region of Alg13. Amino acids represented on the back face of the C-terminal region are involved in the interaction with the Alg14 in the binding pocket.

FIGURE 5.

Sequence alignment of the C terminus of Alg14 from fungi, plants and mammals. A, yeast Alg14 protein was used as query for PSI-BLAST (26) analysis against the UniRef90 protein dataset at UniProt. The last ten C-terminal amino acids of Alg14 orthologues selected from the PSI-BLAST results were manually aligned. The predicted β-strands are shaded in gray, and the glycine residue that is conserved in all Alg14 orthologues is marked with a star. The last three amino acids were boxed.

FIGURE 6.

The hydrophobicity of Alg14 C-terminal amino acids is required for the formation of an activity Alg13/14 complex. Alg14 proteins with the last two or three residues that were replaced by glycine were tested for activity. The P_GAL1-ALG14 strain (XGY151) was transformed with plasmids containing HA-ALG14 (pXG202), HA-alg14-L236G, V237G (pSA13), or HA-alg14-I235G, L236G, V237G (pSA14), and streaked onto YPA plates supplemented with glucose (left panel) and galactose (right panel). Cells were incubated for 2 days at 30 °C.

FIGURE 7.

The N-terminal β-strand of yeast Alg13 is required for viability and protein stability. A, N-terminal sequence of yeast Alg13 protein. The predicted β-strand region is boxed. B, the P_GAL1-ALG13 strain (XGY154) was transformed with plasmids containing ALG13-FLAG (pXG208), alg13-NΔ6-FLAG (pSA1), or alg13-NΔ10-FLAG (pSA2) and streaked onto YPA plates supplemented with galactose (right panel) or glucose (left panel). Cells were grown for 2 days at 30 °C. C, whole cell detergent extracts were prepared from wild-type yeast (W303a) containing plasmids that encode triple FLAG-tagged Alg13p (pXG208), alg13-NΔ6p (pSA1), or alg13-NΔ10p (pSA2). Equivalent amounts of protein in each sample were separated by SDS-PAGE, immunoblotted, and detected with anti-FLAG monoclonal antibody conjugated with alkaline phosphatase as described under “Experimental Procedures.”