The components of the Saccharomyces cerevisiae mannosyltransferase complex M-Pol I have distinct functions in mannan synthesis

The yeast Saccharomyces cerevisiae processes N-linked glycans in the Golgi apparatus in two different ways. Whereas most of the proteins of internal membranes receive a simple core-type structure, a long branched polymer termed mannan is attached to the glycans of many of the proteins destined for the cell wall. The first step in mannan synthesis is the initiation and extension of an alpha-1,6-linked polymannose backbone. This requires the sequential action of two enzyme complexes, mannan polymerases (M-Pol) I and II. M-Pol I contains the proteins Mnn9p and Van1p, although the stoichiometry and individual contributions to enzyme action are unclear. We report here that the two proteins are each present as a single copy in the complex. Both proteins contain a DXD motif found in the active site of many glycosyltransferases, and mutations in this motif in Mnn9p or Van1p reveal that both proteins contribute to mannose polymerization. However, the effects of these mutations on both the in vivo and in vitro activity are distinct, suggesting that the two proteins may have different roles in the complex. Finally, we show that a simple glycoprotein based on hen egg lysozyme can be used as a substrate for modification by purified M-Pol I in vitro.     

The Mnn2 Mannosyltransferase Family Modulates Mannoprotein Fibril Length,Immune Recognition and Virulence of Candida albicans

The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs) recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26). Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans.     

Multi-protein complexes in the cis Golgi of Saccharomyces cerevisiae with alpha-1,6-mannosyltransferase activity.

Anp1p, Van1p and Mnn9p constitute a family of membrane proteins required for proper Golgi function in Saccharomyces cerevisiae. We demonstrate that these proteins colocalize within the cis Golgi, and that they are physically associated in two distinct complexes, both of which contain Mnn9p. Furthermore, we identify two new proteins in the Anp1p-Mnn9p-containing complex which have homology to known glycosyltransferases. Both protein complexes have alpha-1, 6-mannosyltransferase activity, forming a series of poly-mannose structures. These reaction products also contain some alpha-1, 2-linked mannose residues. Our data suggest that these two multi-protein complexes are responsible for the synthesis and initial branching of the long alpha-1,6-linked backbone of the hypermannose structure attached to many yeast glycoproteins.     

A Single Arabinan Chain Is Attached to the Phosphatidylinositol Mannosyl Core of the Major Immunomodulatory Mycobacterial Cell Envelope Glycoconjugate,Lipoarabinomannan

Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5–7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10–12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl “primer.” Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.     

The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex

In the yeast Saccharomyces cerevisiae many of the N-linked glycans on cell wall and periplasmic proteins are modified by the addition of mannan, a large mannose-containing polysaccharide. Mannan comprises a backbone of approximately 50 alpha-1,6-linked mannoses to which are attached many branches consisting of alpha-1,2-linked and alpha-1,3-linked mannoses. The initiation and subsequent elongation of the mannan backbone is performed by two complexes of proteins in the cis Golgi. In this study we show that the product of the MNN10/BED1 gene is a component of one of these complexes, that which elongates the backbone. Analysis of interactions between the proteins in this complex shows that Mnn10p, and four previously characterized proteins (Anp1p, Mnn9p, Mnn11p, and Hoc1p) are indeed all components of the same large structure. Deletion of either Mnn10p, or its homologue Mnn11p, results in defects in mannan synthesis in vivo, and analysis of the enzymatic activity of the complexes isolated from mutant strains suggests that Mnn10p and Mnn11p are responsible for the majority of the alpha-1, 6-polymerizing activity of the complex.     

Hepatocyte Nuclear Factor 4 Provokes Expression of Epithelial Marker Genes, Acting As a Morphogen in Dedifferentiated Hepatoma Cells

The yeast α-1,3-mannosyltransferase (Mnn1p) is localized to the Golgi by independent transmembrane and lumenal domain signals. The lumenal domain is localized to the Golgi complex when expressed as a soluble form (Mnn1-s) by exchange of its transmembrane domain for a cleavable signal sequence (Graham, T. R., and V. A. Krasnov. 1995. Mol. Biol. Cell. 6:809–824). Mutants that failed to retain the lumenal domain in the Golgi complex, called lumenal domain retention (ldr) mutants, were isolated by screening mutagenized yeast colonies for those that secreted Mnn1-s. Two genes were identified by this screen, HOG1, a gene encoding a mitogen-activated protein kinase (MAPK) that functions in the high osmolarity glycerol (HOG) pathway, and LDR1. We have found that basal signaling through the HOG pathway is required to localize Mnn1-s to the Golgi in standard osmotic conditions. Mutations in HOG1 and LDR1 also perturb localization of intact Mnn1p, resulting in its loss from early Golgi compartments and a concomitant increase of Mnn1p in later Golgi compartments.     

Domain Interactions Control Complex Formation and Polymerase Specificity in the Biosynthesis of the Escherichia coli O9a Antigen

The Escherichia coli O9a O-polysaccharide (O-PS) is a prototype for bacterial glycan synthesis and export by an ATP-binding cassette transporter-dependent pathway. The O9a O-PS possesses a tetrasaccharide repeat unit comprising two α-(1→2)- and two α-(1→3)-linked mannose residues and is extended on a polyisoprenoid lipid carrier by the action of a polymerase (WbdA) containing two glycosyltransferase active sites. The N-terminal domain of WbdA possesses α-(1→2)-mannosyltransferase activity, and we demonstrate in this study that the C-terminal domain is an α-(1→3)-mannosyltransferase. Previous studies established that the size of the O9a polysaccharide is determined by the chain-terminating dual kinase/methyltransferase (WbdD) that is tethered to the membrane and recruits WbdA into an active enzyme complex by protein-protein interactions. Here, we used bacterial two-hybrid analysis to identify a surface-exposed α-helix in the C-terminal mannosyltransferase domain of WbdA as the site of interaction with WbdD. However, the C-terminal domain was unable to interact with WbdD in the absence of its N-terminal partner. Through deletion analysis, we demonstrated that the α-(1→2)-mannosyltransferase activity of the N-terminal domain is regulated by the activity of the C-terminal α-(1→3)-mannosyltransferase. In mutants where the C-terminal catalytic site was deleted but the WbdD-interaction site remained, the N-terminal mannosyltransferase became an unrestricted polymerase, creating a novel polymer comprising only α-(1→2)-linked mannose residues. The WbdD protein therefore orchestrates critical localization and coordination of activities involved in chain extension and termination. Complex domain interactions are needed to position the polymerase components appropriately for assembly into a functional complex located at the cytoplasmic membrane.     

Engineering of the Yeast Yarrowia lipolytica for the Production of Glycoproteins Lacking the Outer-Chain Mannose Residues of N-Glycans

In an attempt to engineer a Yarrowia lipolytica strain to produce glycoproteins lacking the outer-chain mannose residues of N-linked oligosaccharides, we investigated the functions of the OCH1 gene encoding a putative α-1,6-mannosyltransferase in Y. lipolytica. The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of YlOCH1 and the lack of in vitro α-1,6-mannosyltransferase activity in the Yloch1 null mutant indicated that YlOCH1 is a functional ortholog of S. cerevisiae OCH1. The oligosaccharides assembled on two secretory glycoproteins, the Trichoderma reesei endoglucanase I and the endogenous Y. lipolytica lipase, from the Yloch1 null mutant contained a single predominant species, the core oligosaccharide Man₈GlcNAc₂, whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man₈GlcNAc₂ to Man₁₂GlcNAc₂. Digestion with α-1,2- and α-1,6-mannosidase of the oligosaccharides from the wild-type and Yloch1 mutant strains strongly supported the possibility that the Yloch1 mutant strain has a defect in adding the first α-1,6-linked mannose to the core oligosaccharide. Taken together, these results indicate that YlOCH1 plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in Y. lipolytica. Therefore, the Yloch1 mutant strain can be used as a host to produce glycoproteins lacking the outer-chain mannoses and further developed for the production of therapeutic glycoproteins containing human-compatible oligosaccharides.     

Domain Organization of the Polymerizing Mannosyltransferases Involved in Synthesis of the Escherichia coli O8 and O9a Lipopolysaccharide O-antigens

The Escherichia coli O9a and O8 polymannose O-polysaccharides (O-PSs) serve as model systems for the biosynthesis of bacterial polysaccharides by ATP-binding cassette transporter-dependent pathways. Both O-PSs contain a conserved primer-adaptor domain at the reducing terminus and a serotype-specific repeat unit domain. The repeat unit domain is polymerized by the serotype-specific WbdA mannosyltransferase. In serotype O9a, WbdA is a bifunctional α-(1→2)-, α-(1→3)-mannosyltransferase, and its counterpart in serotype O8 is trifunctional (α-(1→2), α-(1→3), and β-(1→2)). Little is known about the detailed structures or mechanisms of action of the WbdA polymerases, and here we establish that they are multidomain enzymes. WbdA^O9a contains two separable and functionally active domains, whereas WbdA^O8 possesses three. In WbdC^O9a and WbdB^O9a, substitution of the first Glu of the EX₇E motif had detrimental effects on the enzyme activity, whereas substitution of the second had no significant effect on activity in vivo. Mutation of the Glu residues in the EX₇E motif of the N-terminal WbdA^O9a domain resulted in WbdA variants unable to synthesize O-PS. In contrast, mutation of the Glu residues in the motif of the C-terminal WbdA^O9a domain generated an enzyme capable of synthesizing an altered O-PS repeat unit consisting of only α-(1→2) linkages. In vitro assays with synthetic acceptors unequivocally confirmed that the N-terminal domain of WbdA^O9a possesses α-(1→2)-mannosyltransferase activity. Together, these studies form a framework for detailed structure-function studies on individual domains and a strategy applicable for dissection and analysis of other multidomain glycosyltransferases.     

Glycoside hydrolase from the GH76 family indicates that marine Salegentibacter sp. Hel_I_6 consumes alpha-mannan from fungi

Microbial glycan degradation is essential to global carbon cycling. The marine bacterium Salegentibacter sp. Hel_I_6 (Bacteroidota) isolated from seawater off Helgoland island (North Sea) contains an α-mannan inducible gene cluster with a GH76 family endo-α-1,6-mannanase (ShGH76). This cluster is related to genetic loci employed by human gut bacteria to digest fungal α-mannan. Metagenomes from the Hel_I_6 isolation site revealed increasing GH76 gene frequencies in free-living bacteria during microalgae blooms, suggesting degradation of α-1,6-mannans from fungi. Recombinant ShGH76 protein activity assays with yeast α-mannan and synthetic oligomannans showed endo-α-1,6-mannanase activity. Resolved structures of apo-ShGH76 (2.0 Å) and of mutants co-crystalized with fungal mannan-mimicking α-1,6-mannotetrose (1.90 Å) and α-1,6-mannotriose (1.47 Å) retained the canonical (α/α)₆ fold, despite low identities with sequences of known GH76 structures (GH76s from gut bacteria: <27%). The apo-form active site differed from those known from gut bacteria, and co-crystallizations revealed a kinked oligomannan conformation. Co-crystallizations also revealed precise molecular-scale interactions of ShGH76 with fungal mannan-mimicking oligomannans, indicating adaptation to this particular type of substrate. Our data hence suggest presence of yet unknown fungal α-1,6-mannans in marine ecosystems, in particular during microalgal blooms.Subject terms: Metagenomics, Microbial ecology, Structural biology, Fungal ecology, Molecular ecology     

Interaction Among the Subunits of Golgi Membrane Mannosyltransferase Complexes of the Yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae Mnn9 protein is a type II Golgi membrane protein which concerns in protein mannosylation. When solubilized by Triton X-100, it was recovered in two distinct complexes both having mannosyltransferase activity; one with Van1 protein (V-complex) and the other with Anp1, Hoc1, Mnn10, and Mnn11 proteins (A-complex). Characterization of the null mutants suggested that A-complex is also concerned in protein O-glycosylation. A-complex was more resistant than V-complex to dissociating conditions. Interaction between the lumenal domains of Van1 and Mnn9 was detected by a two-hybrid experiment. The anchor domain of Mnn9 protein could be replaced with other membrane anchors without losing the ability to form complexes similar to V- and A-complexes. Thus the lumenal domains are important to assemble these distinct complexes.     

Crystal Structure and Characterization of the Glycoside Hydrolase Family 62 α-l-Arabinofuranosidase from Streptomyces coelicolor

α-l-Arabinofuranosidase, which belongs to the glycoside hydrolase family 62 (GH62), hydrolyzes arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several α-l-arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-l-arabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N terminus and a catalytic domain at its C terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed β-propeller consisting of five radially oriented anti-parallel β-sheets. Sugar complex structures with l-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an l-arabinose-binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and the catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp²⁰² and Glu³⁶¹ were catalytic residues, and Trp²⁷⁰, Tyr⁴⁶¹, and Asn⁴⁶² were involved in the substrate-binding site for discriminating the substrate structures. In particular, hydrogen bonding between Asn⁴⁶² and xylose at the nonreducing end subsite +2 was important for the higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.     

Unraveling the Novel Structure and Biosynthetic Pathway of O-Linked Glycans in the Golgi Apparatus of the Human Pathogenic Yeast Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated basidiomycete causing cryptococcosis in immunocompromised humans. The cell surface mannoproteins of C. neoformans were reported to stimulate the host T-cell response and to be involved in fungal pathogenicity; however, their O-glycan structure is uncharacterized. In this study, we performed a detailed structural analysis of the O-glycans attached to cryptococcal mannoproteins using HPLC combined with exoglycosidase treatment and showed that the major C. neoformans O-glycans were short manno-oligosaccharides that were connected mostly by α1,2-linkages but connected by an α1,6-linkage at the third mannose residue. Comparison of the O-glycan profiles from wild-type and uxs1Δ mutant strains strongly supports the presence of minor O-glycans carrying a xylose residue. Further analyses of C. neoformans mutant strains identified three mannosyltransferase genes involved in O-glycan extensions in the Golgi. C. neoformans KTR3, the only homolog of the Saccharomyces cerevisiae KRE2/MNT1 family genes, was shown to encode an α1,2-mannosyltransferase responsible for the addition of the second mannose residue via an α1,2-linkage to the major O-glycans. C. neoformans HOC1 and HOC3, homologs of the Saccharomyces cerevisiae OCH1 family genes, were shown to encode α1,6-mannosyltransferases that can transfer the third mannose residue, via an α1,6-linkage, to minor O-glycans containing xylose and to major O-glycans without xylose, respectively. Moreover, the C. neoformans ktr3Δ mutant strain, which displayed increased sensitivity to SDS, high salt, and high temperature, showed attenuated virulence in a mouse model of cryptococcosis, suggesting that the extended structure of O-glycans is required for cell integrity and full pathogenicity of C. neoformans.     

Caenorhabditis elegans N-glycan Core β-galactoside Confers Sensitivity towards Nematotoxic Fungal Galectin CGL2

The physiological role of fungal galectins has remained elusive. Here, we show that feeding of a mushroom galectin, Coprinopsis cinerea CGL2, to Caenorhabditis elegans inhibited development and reproduction and ultimately resulted in killing of this nematode. The lack of toxicity of a carbohydrate-binding defective CGL2 variant and the resistance of a C. elegans mutant defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity depends on the interaction between the galectin and a fucose-containing glycoconjugate. A screen for CGL2-resistant worm mutants identified this glycoconjugate as a Galβ1,4Fucα1,6 modification of C. elegans N-glycan cores. Analysis of N-glycan structures in wild type and CGL2-resistant nematodes confirmed this finding and allowed the identification of a novel putative glycosyltransferase required for the biosynthesis of this glycoepitope. The X-ray crystal structure of a complex between CGL2 and the Galβ1,4Fucα1,6GlcNAc trisaccharide at 1.5 Å resolution revealed the biophysical basis for this interaction. Our results suggest that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms.     

Loss of the Plasma Membrane-Bound Protein Gas1p in Saccharomyces cerevisiae Results in the Release of β1,3-Glucan into the Medium and Induces a Compensation Mechanism To Ensure Cell Wall Integrity

Deletion of GAS1/GGP1/CWH52 results in a lower β-glucan content of the cell wall and swollen, more spherical cells (L. Popolo, M. Vai, E. Gatti, S. Porello, P. Bonfante, R. Balestrini, and L. Alberghina, J. Bacteriol. 175:1879–1885, 1993; A. F. J. Ram, S. S. C. Brekelmans, L. J. W. M. Oehlen, and F. M. Klis, FEBS Lett. 358:165–170, 1995). We show here that gas1Δ cells release β1,3-glucan into the medium. Western analysis of the medium proteins with β1,3-glucan- and β1,6-glucan-specific antibodies showed further that at least some of the released β1,3-glucan was linked to protein as part of a β1,3-glucan–β1,6-glucan–protein complex. These data indicate that Gas1p might play a role in the retention of β1,3-glucan and/or β-glucosylated proteins. Interestingly, the defective incorporation of β1,3-glucan in the cell wall was accompanied by an increase in chitin and mannan content in the cell wall, an enhanced expression of cell wall protein 1 (Cwp1p), and an increase in β1,3-glucan synthase activity, probably caused by the induced expression of Fks2p. It is proposed that the cell wall weakening caused by the loss of Gas1p induces a set of compensatory reactions to ensure cell integrity.     

The N-Linked Outer Chain Mannans and the Dfg5p and Dcw1p Endo-α-1,6-Mannanases Are Needed for Incorporation of Candida albicans Glycoproteins into the Cell Wall

A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-α-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p α-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p α-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix.     

Presenilin Transmembrane Domain 8 Conserved AXXXAXXXG Motifs Are Required for the Activity of the γ-Secretase Complex

Understanding the molecular mechanisms controlling the physiological and pathological activity of γ-secretase represents a challenging task in Alzheimer disease research. The assembly and proteolytic activity of this enzyme require the correct interaction of the 19 transmembrane domains (TMDs) present in its four subunits, including presenilin (PS1 or PS2), the γ-secretase catalytic core. GXXXG and GXXXG-like motifs are critical for TMDs interactions as well as for protein folding and assembly. The GXXXG motifs on γ-secretase subunits (e.g. APH-1) or on γ-secretase substrates (e.g. APP) are known to be involved in γ-secretase assembly and in Aβ peptide production, respectively. We identified on PS1 and PS2 TMD8 two highly conserved AXXXAXXXG motifs. The presence of a mutation causing an inherited form of Alzheimer disease (familial Alzheimer disease) in the PS1 motif suggested their involvement in the physiopathological configuration of the γ-secretase complex. In this study, we targeted the role of these motifs on TMD8 of PSs, focusing on their role in PS assembly and catalytic activity. Each motif was mutated, and the impact on complex assembly, activity, and substrate docking was monitored. Different amino acid substitutions on the same motif resulted in opposite effects on γ-secretase activity, without affecting the assembly or significantly impairing the maturation of the complex. Our data suggest that AXXXAXXXG motifs in PS TMD8 are key determinants for the conformation of the mature γ-secretase complex, participating in the switch between the physiological and pathological functional conformations of the γ-secretase.     

MNN5 encodes an iron-regulated alpha-1,2-mannosyltransferase important for protein glycosylation, cell wall integrity, morphogenesis, and virulence in Candida albicans

The cell walls of microbial pathogens mediate physical interactions with host cells and hence play a key role in infection. Mannosyltransferases have been shown to determine the cell wall properties and virulence of the pathogenic fungus Candida albicans. We previously identified a C. albicans alpha-1,2-mannosyltransferase, Mnn5, for its novel ability to enhance iron usage in Saccharomyces cerevisiae. Here we have studied the enzymatic properties of purified Mnn5 and characterized its function in its natural host. Mnn5 catalyzes the transfer of mannose to both alpha-1,2- and alpha-1,6-mannobiose, and this activity requires Mn2+ as a cofactor and is regulated by the Fe2+ concentration. An mnn5Delta mutant showed a lowered ability to extend O-linked, and possibly also N-linked, mannans, hypersensitivity to cell wall-damaging agents, and a reduction of cell wall mannosylphosphate content, phenotypes typical of many fungal mannosyltransferase mutants. The mnn5Delta mutant also exhibited some unique defects, such as impaired hyphal growth on solid media and attenuated virulence in mice. An unanticipated phenotype was the mnn5Delta mutant's resistance to killing by the iron-chelating protein lactoferrin, rendering it the first protein found that mediates lactoferrin killing of C. albicans. In summary, MNN5 deletion impairs a wide range of cellular events, most likely due to its broad substrate specificity. Of particular interest was the observed role of iron in regulating the enzymatic activity, suggesting an underlying relationship between Mnn5 activity and cellular iron homeostasis.     

Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins

Endoplasmic reticulum–synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2β are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2ΔNLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins.