MNN14 (systematic name YJR061W) encodes a 935-residue single-pass type II membrane protein of the Golgi apparatus in Saccharomyces cerevisiae. It is a paralog of MNN4 and belongs to the fukutin-related/MNN-like family (InterPro IPR009644; PANTHER PTHR15407). MNN14 is a mannosylphosphorylation enzyme: recombinant soluble MNN14 transfers mannosyl-phosphate from GDP-mannose to high-mannose N-glycans (e.g. Man8GlcNAc2 and the Man7-9GlcNAc2 glycans of a therapeutic protein), producing mono- and bis-mannosyl-phosphorylated glycans in an Mn2+-dependent reaction. It acts partially redundantly with MNN4 in vivo: single deletions leave residual mannosylphosphate, whereas simultaneous deletion of MNN4 and MNN14 abolishes N-glycan mannosylphosphorylation. Mannosylphosphorylation adds mannose-1-phosphate to the mannans of fungal glycoproteins, contributing negative surface charge, and is fungal-specific, so loss of MNN4 plus MNN14 is used in yeast glyco-engineering to make human-compatible glycoproteins. The protein carries a conserved DXD motif (498-500) typical of divalent-cation-dependent transferases. The in-vivo division of labour between the intrinsic catalytic activity of MNN14 and MNN4 (curated as a regulator of the Mnn6/Ktr6 mannosylphosphate transferase), the acceptor-position specificity of MNN14, and the basis of the MNN4/MNN14 redundancy remain to be resolved.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0009101 glycoprotein biosynthetic process | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT IBA from PTN001034988 in the PTHR15407 fukutin-related/MNN-like tree. Current PAINT places GO:0009101 at the opisthokont node seeded by Candida, yeast MNN4/MNN14, mouse Fktn and human FKTN annotations. MNN14 acts in N-glycan mannosylphosphorylation, which adds mannose-1-phosphate to glycoprotein-linked mannans and is part of glycoprotein biosynthesis, so the BP is correct. It is more general than the experimentally supported N-glycan-processing annotation below and is retained as a valid but non-core parent process. Reason: Correct but generic BP consistent with the gene's role; superseded for specificity by the experimental GO:0006491 annotation. MNN14's own experimental SGD annotation is expected descendant support for the IBD, not circularity. Retained per GO conventions as a valid IBA inference. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN001034988 · PTN001034988 SUPPORTS TRANSFER Current PAINT places GO:0009101 at this PTHR15407 node. The node is broad, but its glycoprotein-biosynthetic-process assertion is consistent with the experimentally defined mannosylphosphorylation roles of S. cerevisiae MNN14 and MNN4 and with the broader FKTN/ribitol-phosphate side of the family. Supporting Evidence: PMID:28101612 essential for N-glycan mannosylphosphorylation |
| GO:0000139 Golgi membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Subcellular-location annotation supported by the UniProt SUBCELLULAR LOCATION (Golgi apparatus membrane) and by the protein's single-pass type II membrane topology (cytoplasmic 1-21; signal-anchor TM 22-42; lumenal 43-935). Golgi localization is expected for an enzyme/regulator acting on outer-chain mannan maturation and is consistent with the MNN4 paralog. Reason: Domain/topology- and SubCell-supported cellular component; the correct and informative localization for this Golgi mannosylphosphorylation factor. Supporting Evidence: UniProt:P40355 Golgi apparatus membrane UniProt:P40355 Signal-anchor for type II membrane protein |
| GO:0006491 N-glycan processing | IGI PMID:28101612 Abolishment of N-glycan mannosylphosphorylation in glyco-eng... | ACCEPT | Summary: Experimental (IGI) annotation curated by SGD from the genetic interaction with MNN4 (with/from SGD:S000001684). Kim et al. 2017 showed that MNN14 is required for full N-glycan mannosylphosphorylation and that the MNN4+MNN14 double deletion eliminates it, placing MNN14 in N-glycan outer-chain maturation. GO:0006491 (conversion of N-linked glycan to a mature form by glycosidases/glycosyltransferases) appropriately captures this outer-chain modification step. This is the best-supported process annotation and the core biological role. Reason: Experimentally grounded (IGI) core biological process directly supported by the double-deletion phenotype; the single defensible functional placement for this otherwise dark gene. Supporting Evidence: PMID:28101612 essential for N-glycan mannosylphosphorylation PMID:28101612 Double disruption of MNN4 and MNN14 genes was enough to eliminate N-glycan |
| GO:0003674 molecular_function | ND GO_REF:0000015 | REMOVE | Summary: Root-level placeholder assigned by SGD with the ND (No biological Data) code, reflecting that no molecular function had been curated for MNN14. This is now superseded by the dedicated NEW GO:0000031 mannosylphosphate transferase activity row, which carries the direct PMID:33144549 in-vitro biochemical evidence for recombinant soluble MNN14. Reason: The ND placeholder is no longer accurate and should be removed now that the same review asserts the experimentally supported core molecular function as a NEW GO:0000031 row with its own IDA-equivalent PMID:33144549 attribution. Supporting Evidence: PMID:33144549 in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein PMID:33144549 2 mM GDP-mannose (donor substrate) |
| GO:0000031 mannosylphosphate transferase activity | IDA PMID:33144549 In Vitro N-Glycan Mannosyl-Phosphorylation of a Therapeutic ... | NEW | Summary: Proposed NEW molecular-function annotation not currently in GOA. Recombinant soluble MNN14 (rMnn14 77-935) directly transfers mannosyl-phosphate from the donor GDP-mannose to high-mannose N-glycan acceptors, producing mono- and bis-mannosyl-phosphorylated glycans in an Mn2+-dependent reaction optimal at pH 7.5 / 30 C (Kang et al. 2021). GO:0000031 exactly matches this reaction (GDP-mannose + mannose-acceptor -> phosphorylated glycan + GMP) and is the intrinsic catalytic activity of MNN14. Same-host P. pastoris expression tests recovered Mnn14 clones with mannosyl-phosphorylation activity but not YlMpo1 clones, and the activity tracked Mnn14 construct boundaries and His-tag placement. Reason: Experimentally supported (in-vitro biochemical, IDA-equivalent) molecular function for MNN14 that is missing from GOA; this is the core catalytic function of the gene product. Supporting Evidence: PMID:33144549 a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans PMID:33144549 a recombinant Mnn14 protein derived from Saccharomyces cerevisiae PMID:33144549 the main MPEs in Saccharomyces cerevisiae PMID:33144549 P. pastoris clones that secreted Mnn14 with mannosyl-phosphorylation activity were found, whereas clones that secreted YlMpo1 were not found. |
| GO:0005575 cellular_component | ND GO_REF:0000015 | ACCEPT | Summary: Root cellular component ND annotation: when it was made, no cellular component had been curated for this gene product from literature. Reason: ACCEPT under the ND acceptance rule (projects/NO_DATA.md). The evidence this review cites for the cellular component is inferred (from orthologs, family membership or sequence features) or comes from other species; none of it is literature on this gene product in this species. This was judged from the review's own content during the NO_DATA re-action pass, not from a new literature search. Earlier action: REMOVE. Re-checked 2026-10-31: the Mnn14 papers cited in core_functions (PMID:33144549, PMID:28101612) assay its activity, not its localization. |
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Download this section (compressed HTML)Q: Given that MNN14 has intrinsic mannosylphosphate transferase activity in vitro while MNN4 is curated as a regulator of Mnn6/Ktr6, how are catalysis and regulation partitioned between MNN14 and MNN4 in vivo?
Q: Does the conserved DXD motif of MNN14 support catalysis, and is it required for MNN14 function in vivo?
Experiment: Introduce DXD-motif catalytic point mutations into chromosomal MNN14 (and MNN4) and score the N-glycan mannosylphosphate profile in single and double mutants; complement mnn4 mnn14 double deletions with wild-type versus DXD-mutant alleles; and test whether MNN14/MNN4 stimulate purified Mnn6/Ktr6 activity in a reconstituted reaction.
Hypothesis: The DXD motif is required for the in-vivo mannosylphosphate transferase activity of MNN14, and MNN14 contributes catalysis (not only regulation) to cellular mannosylphosphorylation.
Type: structure-function / reconstitution
Experiment: Perform quantitative N-glycan and mannan structural analysis (mass spectrometry) of cell-wall mannoproteins and a secreted reporter glycoprotein from wild-type, mnn4, mnn14, and mnn4 mnn14 strains to map which mannosylphosphate positions depend on each paralog.
Hypothesis: MNN14 and MNN4 act on distinct acceptor positions or substrate classes, explaining their partial redundancy.
Type: comparative glycomics
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The in-vivo division of labour between MNN14 and its paralog MNN4 is unresolved. MNN14 has intrinsic mannosylphosphate transferase activity in vitro, yet MNN4 is curated as a positive regulator (enzyme activator) of the Mnn6/Ktr6 mannosylphosphate transferase, not a catalyst. It is unknown whether, in the cell, MNN14 acts primarily as a catalyst, MNN4 primarily as a regulator, and how the two models are reconciled for two closely related paralogs.
NARROWING BIOLOGY RESIDUAL_SUBGAP
What is known: Firmly established: recombinant soluble MNN14 catalyzes mannosyl-phosphate transfer from GDP-mannose to high-mannose N-glycans in vitro (Mn2+-dependent), so MNN14 has intrinsic transferase activity (GO:0000031); and genetically both MNN4 and MNN14 are required for full N-glycan mannosylphosphorylation. Also established, classically, is that Mnn6/Ktr6 (KRE2/MNT1 family) is a mannosylphosphate transferase and that MNN4 is a positive regulator whose transcript/protein level is rate-limiting; MNN4's curated molecular function is enzyme activator activity (GO:0008047).
Significance: Clarifying whether MNN14 (and MNN4) contribute catalysis, regulation, or both in vivo would resolve an apparent contradiction between the in-vitro enzymology and the classical regulator model, and would sharpen the mannosylphosphorylation pathway model that underpins yeast glyco-engineering.
What would resolve it: In-vivo structure-function analysis (DXD-motif catalytic mutants of MNN14 and MNN4 assayed for the glycan phenotype), reconstitution tests of whether MNN14/MNN4 stimulate Mnn6/Ktr6, and quantitative comparison of the catalytic contribution of each paralog in single and double mutants.
Provenance (the field's own admissions):
Gap: The acceptor-position specificity of MNN14 in vivo is unknown: which mannose residues/positions on N-linked (and possibly O-linked) glycans MNN14 phosphorylates in the cell, and whether MNN4 and MNN14 act on the same or different positions.
OPEN BIOLOGY MF_DARK
What is known: In vitro, MNN14 mannosyl-phosphorylates high-mannose N-glycans (Man8GlcNAc2 and Man7-9GlcNAc2 on rhGAA), producing mono- and bis-mannosyl-phosphorylated products; genetically, N-glycan mannosylphosphorylation is abolished only when both MNN4 and MNN14 are deleted. The precise in-vivo acceptor positions and whether the two paralogs are position-selective have not been mapped.
Significance: Mapping acceptor positions would convert the pathway placement into a fully defined molecular reaction and explain why two paralogs are maintained.
What would resolve it: Structural mannan/N-glycan analysis (e.g. by mass spectrometry) of defined acceptors in wild-type versus mnn14 single and mnn4 mnn14 double mutants, combined with in-vitro reactions on candidate acceptor substrates.
Provenance (the field's own admissions):
Gap: The basis of the functional redundancy between MNN14 and MNN4, and any distinct or condition-specific role of MNN14, are unknown. There is no described standalone loss-of-function phenotype for an mnn14 single mutant beyond the residual mannosylphosphate profile.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: Established: single deletion of either MNN4 or MNN14 leaves residual N-glycan mannosylphosphorylation, and only the double deletion eliminates it, so the two paralogs have overlapping but individually non-essential activities. MNN14 is a non-essential gene, and its expression is repressed by RIM101.
Significance: Understanding whether MNN14 and MNN4 differ in acceptor specificity, substrate class (N- vs O-linked; core vs outer chain), or expression context would explain why two paralogs are maintained and would sharpen the mannosylphosphorylation model relevant to glyco-engineering.
What would resolve it: Comparative glycan profiling and genetic-interaction/expression analyses of mnn4 and mnn14 single mutants under varied growth conditions, and tests of RIM101-dependent regulation of MNN14 relative to MNN4.
Provenance (the field's own admissions):
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Download this section (compressed HTML)Protein required for N-glycan mannosylphosphorylation; non-essential gene; transcription repressed by Rm101p; MNN14 has a paralog, MNN4, that arose from the whole genome duplication
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