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.     

Isolation of protein glycosylation mutants in the fission yeast Schizosaccharomyces pombe.

We have isolated mutants in the fission yeast Schizosaccharomyces pombe that are defective in protein glycosylation. A collection of osmotically sensitive mutants was prepared and screened for glycosylation defects using lectin staining as an assay. Mutants singly defective in four glycoprotein synthesis genes (gps1-4) were isolated, all of which bind less galactose-specific lectin. Acid phosphatase and other glycoproteins from the gps mutants have increased electrophoretic mobility, suggesting that these mutants make glycans of reduced size. N-linked glycan analysis revealed that terminal oligosaccharide modification is defective in the gps1 and gps2 mutants. Both mutants synthesize the Man9GlcNAc2 core glycan but have reduced amounts of larger structures. Modified core glycans from gps1 cells have normal amounts of galactose (Gal) residues, but reduced amounts of Man, consistent with a defect in a Golgi mannosyltransferase in this mutant. In contrast, N-linked oligosaccharides from gps2 mutants have much less Gal than wild type, because of reduced levels of the Gal donor, UDP-Gal. This reduction is caused by decreased activity of UDP-glucose 4-epimerase, which synthesizes UDP-Gal. Neither the gps1 or gps2 mutations are lethal, although the cells grow at reduced rates. These findings suggest that S. pombe cells can survive with incompletely glycosylated cell wall glycoproteins. In particular, these results suggest that Gal, which comprises approximately 30% by weight of cell wall glycoprotein glycans, is not crucial for cell growth or survival.     

Man8GlcNAc2糖基化的酿酒酵母菌株的构建

酿酒酵母糖蛋白的N-糖基化经过高尔基体的修饰后形成聚合度约150-200的甘露寡糖,高尔基体N-糖基化的糖基转移酶Mnn1p和Och1p在甘露寡糖的形成过程中起关键作用。通过同源重组置换敲除了酵母中的MNN1和OCH1基因阻断高尔基体N-糖基化修饰,分离纯化了mnn1 och1突变株中的N-糖蛋白,糖酰胺酶PNGaseF酶解释放的N-糖链经过2-氨基吡啶衍生后,利用HPLC和MALDITOF/MS结合的方法分析了突变株糖蛋白上的N-糖链。结果显示mnn1 och1突变株中的糖蛋白的N-糖链为结构单一的糖链,分子量为1794.66,推测为Man₈GlcNAc₂。     

Contribution of N-linked glycans to the conformation and function of intercellular adhesion molecules (ICAMs)

The crystal structures of the glycosylated N-terminal two domains of ICAM-1 and ICAM-2 provided a framework for understanding the role of glycosylation in the structure and function of intercellular adhesion molecules (ICAMs). The most conserved glycans were less flexible in the structures, interacting with protein residues and contributing to receptor folding and expression. The first N-linked glycan in ICAM-2 contacts an exposed tryptophan residue, defining a conserved glycan-W motif critical for the conformation of the integrin binding domain. The absence of this motif in human ICAM-1 exposes regions used in receptor dimerization and rhinovirus recognition. Experiments with soluble molecules having the N-terminal two domains of human ICAMs identified glycans of the high mannose type N-linked to the second domain of the dendritic cell-specific ICAM-grabbing nonintegrin lectin-ligands ICAM-2 and ICAM-3. About 40% of those receptor molecules bear endoglycosidase H sensitive glycans responsible of the lectin binding activity. High mannose glycans were absent in ICAM-1, which did not bind to the lectin, but they appeared in ICAM-1 mutants with additional N-linked glycosylation and lectin binding activity. N-Linked glycosylation regulate both conformation and immune related functions of ICAM receptors.     

Preliminary characterization of Yor180Cp: identification of a novel peroxisomal protein of saccharomyces cerevisiae involved in fatty acid metabolism.

Here we report the preliminary characterization of Yor180Cp, a novel peroxisomal protein involved in fatty acid metabolism in the yeast Saccharomyces cerevisiae. A computer-based screen identified Yor180Cp as a putative peroxisomal protein, and Yor180Cp targeted GFP to peroxisomes in a PEX8-dependent manner. Yor180Cp was also detected by mass spectrometric analysis of an HPLC-separated extract of yeast peroxisomal matrix proteins. YOR180C is upregulated during growth on oleic acid, and deletion of YOR180C from the yeast genome resulted in a mild but significant growth defect on oleic acid, indicating a role for Yor180Cp in fatty acid metabolism. In addition, we observed that yor180cDelta cells fail to efficiently import the enzyme Delta3,Delta2-enoyl-CoA isomerase (Eci1p) to peroxisomes. This result suggested that Yor180Cp might associate with Eci1p in vivo, and a Yor180Cp-Eci1p interaction was detected using the yeast two-hybrid system. Potential roles for Yor180Cp in peroxisomal fatty acid metabolism are discussed.     

Yeast GTB1 encodes a subunit of glucosidase II required for glycoprotein processing in the endoplasmic reticulum

Glucosidase II is essential for sequential removal of two glucose residues from N-linked glycans during glycoprotein biogenesis in the endoplasmic reticulum. The enzyme is a heterodimer whose alpha-subunit contains the glycosyl hydrolase active site. The function of the beta-subunit has yet to be defined, but mutations in the human gene have been linked to an autosomal dominant form of polycystic liver disease. Here we report the identification and characterization of a Saccharomyces cerevisiae gene, GTB1, encoding a polypeptide with 21% sequence similarity to the beta-subunit of human glucosidase II. The Gtb1 protein was shown to be a soluble glycoprotein (96-102 kDa) localized to the endoplasmic reticulum lumen where it was present in a complex together with the yeast alpha-subunit homologue Gls2p. Surprisingly, we found that Deltagtb1 mutant cells were specifically defective in the processing of monoglucosylated glycans. Thus, although Gls2p is sufficient for cleavage of the penultimate glucose residue, Gtb1p is essential for cleavage of the final glucose. Our data demonstrate that Gtb1p is required for normal glycoprotein biogenesis and reveal that the final two glucose-trimming steps in N-glycan processing are mechanistically distinct.     

Structures of the carbohydrate recognition domain of Ca2+-independent cargo receptors Emp46p and Emp47p

Emp46p and Emp47p are type I membrane proteins, which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus by vesicles coated with coat protein complexes I and II (COPI and COPII). They are considered to function as cargo receptors for exporting N-linked glycoproteins from the ER. We have determined crystal structures of the carbohydrate recognition domains (CRDs) of Emp46p and Emp47p of Saccharomyces cerevisiae, in the absence and presence of metal ions. Both proteins fold as a beta-sandwich, and resemble that of the mammalian ortholog, p58/ERGIC-53. However, the nature of metal binding is distinct from that of Ca(2+)-dependent p58/ERGIC-53. Interestingly, the CRD of Emp46p does not bind Ca(2+) ion but instead binds K(+) ion at the edge of a concave beta-sheet whose position is distinct from the corresponding site of the Ca(2+) ion in p58/ERGIC-53. Binding of K(+) ion to Emp46p appears essential for transport of a subset of glycoproteins because the Y131F mutant of Emp46p, which cannot bind K(+) ion fails to rescue the transport in disruptants of EMP46 and EMP47 genes. In contrast the CRD of Emp47p binds no metal ions at all. Furthermore, the CRD of Emp46p binds to glycoproteins carrying high mannosetype glycans and the is promoted by binding not the addition of Ca(2+) or K(+) ion in These results suggest that Emp46p can be regarded as a Ca(2+)-independent intracellular lectin at the ER exit sites.     

YOR129c, a new element interacting with Cnm67p, a component of the spindle pole body of Saccharomyces cerevisiae

The Saccharomyces cerevisiae spindle pole body (SPB) consists of numerous proteins forming the outer, inner and central plaques. The protein Cnm67 is an important component of the outer plaque. The C-terminus of this protein contains a determinant important for its SPB localization. We identified a protein encoded by YOR129c which interacts with this C-terminus in the two-hybrid system. YOR129c and CNM67 exhibit weak genetic interaction. The double deletion strain yor129cdelta cnm67delta exhibits moderately increased resistance to 0.1M LiCl and hygromycin B compared with the cnm67delta single mutant. We propose that the YOR129c protein is an accessory factor associated with the cytoplasmic face of SPB and plays a role in cation homeostasis and/or multidrug resistance.     

N-linked glycosylation of macrophage-derived PAF-AH is a major determinant of enzyme association with plasma HDL

Human plasma PAF-AH (platelet-activating factor-acetylhydrolase) is a Ca(2)+-independent phospholipase A2 of hematopoietic origin associated with LDL and HDL; it degrades PAF and oxidizes phospholipids. We show that human macrophages synthesize PAF-AH as a premedial Golgi precursor containing high mannose N-linked glycans. Secreted PAF-AH possesses a molecular mass of approximately 55 kDa and contains mature N-linked glycans. Secreted PAF-AH activity (90 +/- 4% of the total) bound to a wheat germ lectin column and could be eluted with N-acetylglucosamine, whereas digestion with N-acetylneuraminidase II completely abolished enzyme absorption. Tunicamycin significantly reduced cell-associated PAF-AH activity and inhibited enzyme secretion; but it did not alter the ratio of secreted to cell-associated enzyme (1.8 at 6 h and 3.1 at 24 h), suggesting that glycosylation is not essential for PAF-AH secretion. Digestion of cell-associated PAF-AH or secreted PAF-AH with peptide N-glycosidase F affected neither catalytic activity nor its resistance to proteolysis with trypsin or proteinase K; in addition, it did not affect PAF-AH association with LDL, but significantly increased its association with HDL. We suggest that macrophage-derived PAF-AH contains heterogeneous asparagine-conjugated sugar chain(s) involving sialic acid, which hinders its association with HDL but does not influence the secretion, catalytic activity, or resistance of PAF-AH to proteases.     

The location of asparagine-linked glycans on West Nile virions controls their interactions with CD209 (dendritic cell-specific ICAM-3 grabbing nonintegrin)

Mammalian cell-derived West Nile virus preferentially infects cells expressing the C-type lectin CD209L (dendritic cellspecific ICAM-3 grabbing nonintegrin-related protein; liver- and lymph node-specific ICAM-3 grabbing nonintegrin) but not cells expressing CD209 (dendritic cell-specific ICAM-3 grabbing nonintegrin). In contrast, Dengue virus infection is enhanced in cells expressing either attachment factor. The West Nile virus envelope (E) protein contains a single N-linked glycosylation site at residue 154, whereas Dengue virus E contains sites at residues 153 and 67. We introduced a glycosylation site at position 67 into West Nile virus E. Reporter virus particles pseudotyped with this E protein infected cells using either CD209 or CD209L. We also introduced glycosylation sites at several novel positions. All sites allowed CD209L-mediated infection, but only a subset promoted CD209 use. As seen for other viruses, mannose-rich glycans on West Nile virus were required for its interactions with CD209. Surprisingly, however, mannose-rich glycans were not required for CD209L-mediated infection. Complex glycans, particularly N-acetylglucosamine-terminated structures, were able to mediate reporter virus particle interactions with CD209L. We propose that CD209L recognizes glycosylated flaviviruses with broad specificity, whereas CD209 is selective for flaviviruses bearing mannose-rich glycans. The location of the N-linked glycosylation sites on a virion determines the types of glycans incorporated, thus controlling viral tropism for CD209-expressing cells.     

Functional analysis of the ALG3 gene encoding the Dol-P-Man: Man5GlcNAc2-PP-Dol mannosyltransferase enzyme of P. pastoris

N-glycans are synthesized in both yeast and mammals through the ordered assembly of a lipid-linked core Glc(3)Man(9)GlcNAc(2) structure that is subsequently transferred to a nascent protein in the endoplasmic reticulum. Once folded, glycoproteins are then shuttled to the Golgi, where additional but divergent processing occurs in mammals and fungi. We cloned the Pichia pastoris homolog of the ALG3 gene, which encodes the enzyme that converts Man(5)GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP. Deletion of this gene in an och1 mutant background resulted in the secretion of glycoproteins with a predicted Man(5)GlcNAc(2) structure that could be trimmed to Man(3)GlcNAc(2) by in vitro alpha-1,2-mannosidase treatment. However, several larger glycans ranging from Hex(6)GlcNAc(2) to Hex(12)GlcNAc(2) were also observed that were recalcitrant to an array of mannosidase digests. These results contrast the far simpler glycan profile found in Saccharomyces cerevisiae alg3-1 och1, indicating diverging Golgi processing in these two closely related yeasts. Finally, analysis of the P. pastoris alg3 deletion mutant in the presence and absence of the outer chain initiating Och1p alpha-1,6-mannosyltransferase activity suggests that the PpOch1p has a broader substrate specificity compared to its S. cerevisiae counterpart.     

Identification of a human orthologue of Sec34p as a component of the cis-Golgi vesicle tethering machinery

The roles of the components of the Sec34p protein complex in intracellular membrane trafficking, first identified in the yeast Saccharomyces cerevisiae, have yet to be characterized in higher eukaryotes. We cloned a human cDNA whose predicted amino acid sequence showed 41% similarity to yeast Sec34p with homology throughout the entire coding region. Affinity-purified antibodies raised against the human SEC34 protein (hSec34p) recognized a cellular protein of 94 kDa in both soluble and membrane fractions. Like yeast Sec34p, cytosolic hSec34p migrated with an apparent molecular mass of 300 kDa on a glycerol velocity gradient, suggesting that it is part of a protein complex. Immunofluorescence microscopy localized hSec34p to the Golgi compartment in cells of all species examined, where it co-localized well with the cis/medial Golgi marker membrin and partially co-localized with cis-Golgi network marker p115 and trans-Golgi marker TGN38. The co-localization with membrin was maintained at 15 degrees C and after microtubule depolymerization with nocodazole. During transport of the tsO45 vesicular stomatitis virus G protein through the Golgi, there was significant overlap with the hSec34p compartment. Green fluorescent protein-hSec34 expressed in HeLa cells was restricted to Golgi cisternae, and its membrane association was sensitive to brefeldin A treatment. Taken together, our findings indicate that hSec34p is part of a peripheral membrane protein complex localized on cis/medial Golgi cisternae where it may participate in tethering intra-Golgi transport vesicles.     

Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris

The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin-related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9-11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.     

Protein sorting in the late Golgi of Saccharomyces cerevisiae does not require mannosylated sphingolipids

Glycosphingolipids are widely viewed as integral components of the Golgi-based machinery by which membrane proteins are targeted to compartments of the endosomal/lysosomal system and to the surface domains of polarized cells. The yeast Saccharomyces cerevisiae creates glycosphingolipids by transferring mannose to the head group of inositol phosphorylceramide (IPC), yielding mannosyl-IPC (MIPC). Addition of an extra phosphoinositol group onto MIPC generates mannosyldi-IPC (M(IP)2C), the final and most abundant sphingolipid in yeast. Mannosylation of IPC is partially dependent on CSG1, a gene encoding a putative sphingolipidmannosyltransferase. Here we show that open reading frame YBR161w, renamed CSH1, is functionally homologous to CSG1 and that deletion of both genes abolishes MIPC and M(IP)2C synthesis without affecting protein mannosylation. Csg1p and Csh1p are closely related polytopic membrane proteins that co-localize with IPC synthase in the medial-Golgi. Loss of Csg1p and Csh1p has no effect on clathrin- or AP-3 adaptor-mediated protein transport from the Golgi to the vacuole. Moreover, segregation of the periplasmic enzyme invertase, the plasma membrane ATPase Pma1p and the glycosylphosphatidylinositol-anchored protein Gas1p into distinct classes of secretory vesicles occurs independently of Csg1p and Csh1p. Our results indicate that protein sorting in the late Golgi of yeast does not require production of mannosylated sphingolipids.     

Analysis of folate binding protein N-linked glycans by mass spectrometry

The folate binding protein (FBP), also known as the folate receptor (FR), is a glycoprotein which binds the vitamin folic acid and its analogues. FBP contains multiple N-glycosilation sites, is selectively expressed in tissues and body fluids, and mediates targeted therapies in cancer and inflammatory diseases. Much remains to be understood about the structure, composition, and the tissue specificities of N-glycans bound to FBP. Here, we performed structural characterization of N-linked glycans originating from bovine and human milk FBPs. The N-linked glycans were enzymatically released from FBPs, purified, and permethylated. Native and permethylated glycans were further analyzed by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS), while tandem MS (MS/MS) was used for their structural characterization. The assignment of putative glycan structures from MS and MS/MS data was achieved using Functional Glycomics glycan database and SimGlycan software, respectively. It was found that FBP from human milk contains putative structures that have composition consistent with high-mannose (Hex(5-6)HexNAc(2)) as well as hybrid and complex N-linked glycans (NeuAc(0-1)Fuc(0-3)Hex(3-6)HexNAc(3-5)). The FBP from bovine milk contains putative structures corresponding to high-mannose (Hex(4-9)HexNAc(2)) as well as hybrid and complex N-linked glycans (Hex(3-6)HexNAc(3-6)), but these glycans mostly do not contain fucose and sialic acid. Glycomic characterization of FBP provides valuable insight into the structure of this pharmacologically important glycoprotein and may have utility in tissue-selective drug targeting and as a biomarker.     

Characterization of a glucose-repressed pyruvate kinase (Pyk2p) in Saccharomyces cerevisiae that is catalytically insensitive to fructose-1,6-bisphosphate.

We have characterized the gene YOR347c of Saccharomyces cerevisiae and shown that it encodes a second functional pyruvate kinase isoenzyme, Pyk2p. Overexpression of the YOR347c/PYK2 gene on a multicopy vector restored growth on glucose of a yeast pyruvate kinase 1 (pyk1) mutant strain and could completely substitute for the PYK1-encoded enzymatic activity. PYK2 gene expression is subject to glucose repression. A pyk2 deletion mutant had no obvious growth phenotypes under various conditions, but the growth defects of a pyk1 pyk2 double-deletion strain were even more pronounced than those of a pyk1 single-mutation strain. Pyk2p is active without fructose-1,6-bisphosphate. However, overexpression of PYK2 during growth on ethanol did not cause any of the deleterious effects expected from a futile cycling between pyruvate and phosphoenolpyruvate. The results indicate that the PYK2-encoded pyruvate kinase may be used under conditions of very low glycolytic flux.     

Approaches to the study of N-linked glycoproteins in human plasma using lectin affinity chromatography and nano-HPLC coupled to electrospray linear ion trap--Fourier transform mass spectrometry

In this publication, we will describe the combination of lectin affinity chromatography with nano high performance liquid chromatography (HPLC) coupled to a linear ion trap Fourier transform mass spectrometer (capillary LC-LTQ/FTMS) to characterize N-linked glycosylation structures in human plasma proteins. We used a well-characterized glycoprotein, tissue plasminogen activator (rt-PA), which is present at low levels in blood, as a standard to determine the dynamic range of this approach. N-linked glycopeptides derived from rt-PA could be characterized at a ratio of 1:200 in human plasma (rtPA: total plasma protein, w/w) by accurate mass measurement in the FTMS and fragmentation (MS(n)) in the linear ion trap. We demonstrated that this platform has the potential to characterize the general N-linked glycosylation structures of abundant glycoproteins present in human plasma without the requirement for antibody-based purification, or additional carbohydrate analytical protocols. This conclusion was supported by the determination of carbohydrate structures for three glycoproteins, IgG, haptoglobin, and alpha-1-acid glycoprotein, at their natural levels in a human plasma sample, but only after the lectin enrichment step.     

Differential lectin binding patterns in the oviductal ampulla of the horse during oestrus

We investigated the oligosaccharide sequence of glycoconjugates, mainly sialoglycoconjugates, in the horse oviductal ampulla during oestrus by means of lectin and pre-lectin methods such as the KOH-neuraminidase procedure to remove sialic acid residues and incubation with N-glycosidase F to cleave N-linked glycans. Ciliated cells displayed N-linked oligosaccharides throughout the cytoplasm. The cilia glycocalyx expressed both N- and O-linked (mucin-type) oligosaccharides, both showing a high variety of terminal sequences. In the most non-ciliated cells, the whole cytoplasm contained N-linked oligosaccharides with terminal alphaGal as well as mucin-type glycans with terminal Forssman pentasaccharides. In a few scattered non-ciliated cells, the whole cytoplasm displayed sialylated N-linked oligosaccharides with terminal Neu5Ac-GalNAc and O-linked glycans terminating with neutral and/or alphaGalNAc, Neu5Ac alpha2,6Gal/GalNAc, Neu5AcGal beta1,3GalNAc. Supra-nuclear granules, probably Golgi zones, of non-ciliated cells showed mainly O-linked glycans rich in sialic acid residues. The luminal surface of non-ciliated cells showed N-linked oligosaccharides, containing terminal/internal alphaMan/alphaGlc, betaGlcNAc and terminal alphaGal, as well as mucin-type oligosaccharides terminating with a large variety of either neutral saccharides or sialylated sequences. Apical protrusions containing O-linked oligosaccharides with terminal Forssman pentasaccharide, Neu5Ac-Gal beta1,4GlcNAc, Neu5Ac-GalNAc were seen in non-ciliated cells scattered along the epithelium. These findings show the presence of sialoglycoconjugates in the oviductal ampulla epithelium of the mare and the existence of different lectin binding profiles between ciliated and non-ciliated (secretory) cells, as well as the presence of non-ciliated cell sub-types which might determine functional differences along the ampullary epithelium of mare oviduct.     

The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex

We have obtained and characterized a genomic clone of SEC14, a Saccharomyces cerevisiae gene whose product is required for export of yeast secretory proteins from the Golgi complex. Gene disruption experiments indicated that SEC14 is an essential gene for yeast vegetative growth. Nucleotide sequence analysis revealed the presence of an intron within the SEC14 structural gene, and predicted the synthesis of a hydrophilic polypeptide of 35 kD in molecular mass. In confirmation, immunoprecipitation experiments demonstrated SEC14p to be an unglycosylated polypeptide, with an apparent molecular mass of some 37 kD, that behaved predominantly as a cytosolic protein in subcellular fractionation experiments. These data were consistent with the notion that SEC14p is a cytosolic factor that promotes protein export from yeast Golgi. Additional radiolabeling experiments also revealed the presence of SEC14p-related polypeptides in extracts prepared from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. Furthermore, the K. lactis SEC14p was able to functionally complement S. cerevisiae sec14ts defects. These data suggested a degree of conservation of SEC14p structure and function in these yeasts species.