Characteristics and structural requirements of apical sorting of the rat growth hormone through the O-glycosylated stalk region of intestinal sucrase-isomaltase

The apical sorting of the small intestinal membrane glycoprotein sucrase-isomaltase (SI) depends on the presence of O-linked glycans and the transmembrane domain. Here, we investigate the role of O-glycans carried by the Ser/Thr-rich stalk region of SI as an apical sorting signal and evaluate the spatial requirements for an efficient recognition of this signal. Several hybrid proteins are generated comprising the unsorted and unglycosylated protein, the rat growth hormone (rGH), fused to either the transmembrane domain of SI (GH-SI(TM)), or the transmembrane and the stalk domains (GH-SI(SR/TM)). Both constructs are randomly distributed over the apical and basolateral membranes of MDCK cells indicating that neither the transmembrane domain nor the O-glycans are sufficient per se for an apical delivery. Only when a polyglycine spacer is inserted between the stalk region of SI and the luminal part of rGH in the GH-SI(Gly/SR/TM) fusion protein does efficient apical sorting of an O-glycosylated protein as well as a time-dependent association with detergent-insoluble lipid microdomains occur. Obviously, the polyglycine spacer facilitates the accessibility of the O-glycans in GH-SI(Gly/SR/TM) to a putative sorting receptor, whereas these glycans are inadequately recognized in GH-SI(SR/TM). We conclude that the O-glycans in the stalk region of SI act as an apical sorting signal within a sorting machinery that comprises at least a carbohydrate-binding protein and fulfills specific spatial requirements provided, for example by a polyglycine spacer in the context of rGH or the P-domain within the SI enzyme complex.     

Altered folding, turnover, and polarized sorting act in concert to define a novel pathomechanism of congenital sucrase-isomaltase deficiency

Naturally occurring mutants of membrane and secretory proteins are often associated with the pathogenesis of human diseases. Here, we describe the molecular basis of a novel phenotype of congenital sucrase-isomaltase deficiency (CSID), a disaccharide malabsorption disorder of the human intestine in which several structural features and functional capacities of the brush-border enzyme complex sucrase-isomaltase (SI) are affected. The cDNA encoding SI from a patient with CSID reveals a mutation in the isomaltase subunit of SI that results in the substitution of a cysteine by an arginine at amino acid residue 635 (C635R). When this mutation is introduced into the wild type cDNA of SI a mutant enzyme, SI(C635R), is generated that shows a predominant localization in the endoplasmic reticulum. Nevertheless, a definite localization of SI(C635R) in the Golgi apparatus and at the cell surface could be also observed. Epitope mapping with conformation-specific mAbs protease sensitivity assays, and enzymatic activity measurements demonstrate an altered folding pattern of SI(C635R) that is responsible for a substantially increased turnover rate and an aberrant sorting profile. Thus, SI(C635R) becomes distributed also at the basolateral membrane in contrast to wild type SI. Concomitant with the altered sorting pattern, the partial detergent extractability of wild type SI shifts to a complete detergent solubility with Triton X-100. The mutation has therefore affected an epitope responsible for the apical targeting fidelity of SI. Altogether, the combined effects of the C635R mutation on the turnover rate, function, polarized sorting, and detergent solubility of SI constitute a unique and novel pathomechanism of CSID.     

A glutamine to proline exchange at amino acid residue 1098 in sucrase causes a temperature-sensitive arrest of sucrase-isomaltase in the endoplasmic reticulum and cis-Golgi

A striking feature of phenotype II in congenital sucrase-isomaltase deficiency is the retention of the brush border protein sucrase-isomaltase (SI) in the cis-Golgi. This transport block is the consequence of a glutamine to proline substitution at amino acid residue 1098 of the sucrase subunit. Here we provide unequivocal biochemical and confocal data to show that the SI(Q/P) mutant reveals characteristics of a temperature-sensitive mutant. Thus, correct folding, competent intracellular transport, and full enzymatic activity can be partially restored by expression of the mutant SI(Q/P) at the permissive temperature of 20 degrees C instead of 37 degrees C. The acquisition of normal trafficking and function appears to utilize several cycles of anterograde and retrograde steps between the endoplasmic reticulum and the Golgi implicating the molecular chaperones calnexin and heavy chain-binding protein. The data presented in this communication are to our knowledge the first to implicate a temperature-sensitive mutation in an intestinal enzyme deficiency or an intestinal disorder.     

O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts.

The plasma membrane of polarised epithelial cells is characterised by two structurally and functionally different domains, the apical and basolateral domains. These domains contain distinct protein and lipid constituents that are sorted by specific signals to the correct surface domain [1]. The best characterised apical sorting signal is that of glycophosphatidylinositol (GPI) membrane anchors [2], although N-linked glycans on some secreted proteins [3] and O-linked glycans [4] also function as apical sorting signals. In the latter cases, however, the underlying sorting mechanisms remain obscure. Here, we have analysed the role of O-glycosylation in the apical sorting of sucrase-isomaltase (SI), a highly polarised N- and O-glycosylated intestinal enzyme, and the mechanisms underlying this process. Inhibition of O-glycosylation by benzyl-N-acetyl-alpha-D-galactosaminide (benzyl-GalNAc) was accompanied by a dramatic shift in the sorting of SI from the apical membrane to both membranes. The sorting mechanism of SI involves its association with sphingolipid- and cholesterol-rich membrane rafts because this association was eliminated when O-glycosylation was inhibited by benzyl-GaINAc. The results demonstrate for the first time that O-linked glycans mediate apical sorting through association with lipid rafts.     

Distinct cytoskeletal tracks direct individual vesicle populations to the apical membrane of epithelial cells

A key aspect in the structure of epithelial and neuronal cells is the maintenance of a polarized organization based on highly specific sorting machinery at the exit site of the trans Golgi network (TGN). Epithelial cells sort protein and lipid components into different sets of carriers for the apical or basolateral plasma membrane. The two intestinal proteins lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are delivered to the apical plasma membrane of epithelial cells with high fidelity but differ in their affinity to detergent-insoluble, glycolipid-enriched complexes (DIGs). Using a two-color labeling technique, we have recently characterized two post-Golgi vesicle populations that direct LPH and SI separately to the apical cell surface. Here, we investigated the structure and identification of protein components in these vesicle populations and assessed the role of cytoskeletal post-Golgi transport routes for apical cargo. Apart from the central role of microtubules in vesicle transport, we demonstrate that the transport of SI-carrying apical vesicles (SAVs) occurs along actin tracks in the cellular periphery, whereas LPH-carrying apical vesicles (LAVs) are transferred in an actin-independent fashion to the apical membrane. Our data further indicate that myosin 1A is the actin-associated motor protein that drives SAVs along actin filaments to the apical cell surface.     

Alpha-kinase 1, a new component in apical protein transport

A key aspect in the structure of epithelial cells is the maintenance of a polarized organization based on a highly specific sorting machinery for cargo destined for the apical or the basolateral membrane domain at the exit site of the trans-Golgi network. We could recently identify two distinct post-trans-Golgi network vesicle populations that travel along separate routes to the plasma membrane, a lipid raft-dependent and a lipid raft-independent pathway. A new component of raft-carrying apical vesicles is alpha-kinase 1 (ALPK1), which was identified in immunoisolated vesicles carrying raft-associated sucrase-isomaltase (SI). This kinase was absent from vesicles carrying raft-non-associated lactase-phlorizin hydrolase. The expression of ALPK1 increases by the time of epithelial cell differentiation, whereas the intracellular localization of ALPK1 on apical transport vesicles was confirmed by confocal analysis. A phosphorylation assay on isolated SI-carrying vesicles revealed the phosphorylation of a protein band of about 105 kDa, which could be identified as the motor protein myosin I. Finally, a specific reduction of ALPK1-expression by RNA interference results in a significant decrease in the apical delivery of SI. Taken together, our data suggest that the phosphorylation of myosin I by ALPK1 is an essential process in the apical trafficking of raft-associated SI.     

Correlation of the amino-acid sequence and the 3D structure of the functional domain of EmaA from Aggregatibacter actinomycetemcomitans

Adhesion to collagen is an important virulence determinant for the periodontal pathogen Aggregatibacter actinomycetemcomitans. Binding to collagen is mediated by the extracellular-matrix protein adhesin-A (EmaA). EmaA is a homotrimeric autotransporter protein that forms flexible antenna-like appendages on the bacterium surface. An ellipsoidal structure at the distal end of the appendage, composed of three subdomains, contains the functional domain of the molecule. A correlation between amino-acid sequence and subdomain structure (SI and SII) was proposed based on an analysis of the volume/molecular weight ratio. EmaA from three mutant strains (deletions of amino-acids 70-206 and 70-386 and a substitution mutation G162S) has been studied by electron microscopy to test this hypothesis. 3D structures were analyzed using single-axis tilt tomography of negatively stained preparations of bacteria combined with subvolume averaging. Additionally, a large number of 2D images of the apical domain of the adhesins from the mutants were extracted from micrographs of the bacterial surface, aligned and classified. The combined data showed that amino-acids 70-206 localize to subdomain SI and 70-386 comprise subdomains SI and SII. Moreover, we showed that the substitution mutation G162S, which abolishes collagen binding activity, does not affect the overall structural integrity of the functional domain. However, the structure of subdomain SI in this mutant is slightly altered with respect to the wild-type strain. These data also have allowed us to interpret the architectural features of each subdomain of EmaA in more detail and to correlate the 3D structure of the functional domain of EmaA with the amino-acid sequence.     

Structural determinants required for apical sorting of an intestinal brush-border membrane protein

The distinct protein and lipid constituents of the apical and basolateral membranes in polarized cells are sorted by specific signals. O-Glycosylation of a highly polarized intestinal brush-border protein sucrase isomaltase is implicated in its apical sorting through interaction with sphingolipid-cholesterol microdomains. We characterized the structural determinants required for this mechanism by focusing on two major domains in pro-SI, the membrane anchor and the Ser/Thr-rich stalk domain. Deletion mutants lacking either domain, pro-SI(DeltaST) (stalk-free) and pro-SI(DeltaMA) (membrane anchor-free), were constructed and expressed in polarized Madin-Darby canine kidney cells. In the absence of the membrane anchoring domain, pro-SI(DeltaMA) does not associate with lipid rafts and the mutant is randomly delivered to both membranes. Therefore, the O-glycosylated stalk region is not sufficient per se for the high fidelity of apical sorting of pro-SI. Pro-SI(DeltaST) does not associate either with lipid rafts and its targeting behavior is similar to that of pro-SI(DeltaMA). Only wild type pro-SI containing both determinants, the stalk region and membrane anchor, associates with lipid microdomains and is targeted correctly to the apical membrane. However, not all sequences in the stalk region are required for apical sorting. Only O-glycosylation of a stretch of 12 amino acids (Ala(37)-Pro(48)) juxtapose the membrane anchor is required in conjunction with the membrane anchoring domain for correct targeting of pro-SI to the apical membrane. Other O-glycosylated domains within the stalk (Ala(49)-Pro(57)) are not sufficient for apical sorting. We conclude that the recognition signal for apical sorting of pro-SI comprises O-glycosylation of the Ala(37)-Pro(48) stretch and requires the presence of the membrane anchoring domain.     

Requirement for galectin-3 in apical protein sorting

The central aspect of epithelial cells is their polarized structure, characterized by two distinct domains of the plasma membrane, the apical and the basolateral membrane. Apical protein sorting requires various signals and different intracellular routes to the cell surface. The first apical targeting motif identified is the membrane anchoring of a polypeptide by glycosyl-phosphatidyl-inositol (GPI). A second group of apical signals involves N- and O-glycans, which are exposed to the luminal side of the sorting organelle. Sucrase-isomaltase (SI) and lactase-phlorizin hydrolase (LPH), which use separate transport platforms for trafficking, are two model proteins for the study of apical protein sorting. In contrast to LPH, SI associates with sphingolipid/cholesterol-enriched membrane microdomains or "lipid rafts". After exit form the trans-Golgi network (TGN), the two proteins travel in distinct vesicle populations, SAVs (SI-associated vesicles) and LAVs (LPH-associated vesicles) . Here, we report the identification of the lectin galectin-3 delivering non-raft-dependent glycoproteins in the lumen of LAVs in a carbohydrate-dependent manner. Depletion of galectin-3 from MDCK cells results in missorting of non-raft-dependent apical membrane proteins to the basolateral cell pole. This suggests a direct role of galectin-3 in apical sorting as a sorting receptor.     

Biogenetic pathways of plasma membrane proteins in Caco-2, a human intestinal epithelial cell line

We studied the sorting and surface delivery of three apical and three basolateral proteins in the polarized epithelial cell line Caco-2, using pulse-chase radiolabeling and surface domain-selective biotinylation (Le Bivic, A., F. X. Real, and E. Rodriguez-Boulan. 1989. Proc. Natl. Acad. Sci. USA. 86:9313-9317). While the basolateral proteins (antigen 525, HLA-I, and transferrin receptor) were targeted directly and efficiently to the basolateral membrane, the apical markers (sucrase-isomaltase [SI], aminopeptidase N [APN], and alkaline phosphatase [ALP]) reached the apical membrane by different routes. The large majority (80%) of newly synthesized ALP was directly targeted to the apical surface and the missorted basolateral pool was very inefficiently transcytosed. SI was more efficiently targeted to the apical membrane (greater than 90%) but, in contrast to ALP, the missorted basolateral pool was rapidly transcytosed. Surprisingly, a distinct peak of APN was detected on the basolateral domain before its accumulation in the apical membrane; this transient basolateral pool (at least 60-70% of the enzyme reaching the apical surface, as measured by continuous basal addition of antibodies) was efficiently transcytosed. In contrast with their transient basolateral expression, apical proteins were more stably localized on the apical surface, apparently because of their low endocytic capability in this membrane. Thus, compared with two other well-characterized epithelial models, MDCK cells and the hepatocyte, Caco-2 cells have an intermediate sorting phenotype, with apical proteins using both direct and indirect pathways, and basolateral proteins using only direct pathways, during biogenesis.     

Single point mutations in domain II of the yeast mitochondrial release factor mRF-1 affect ribosome binding.

We have recently described two yeast strains that are mutated in the MRF1 gene encoding the mitochondrial release factor mRF-1. Both mutants provoke gene-specific defects in mitochondrial translational termination. In the present study we report the cloning, sequencing, as well as an analysis of residual activities of both mutant mrf1 alleles. Each allele specifies a different single amino acid substitution located one amino acid apart. The amino acid changes do not affect the level or cellular localization of the mutant proteins, since equal amounts of wild type and mutant mRF-1 were detected in the mitochondrial compartment. Over-expression of the mutant alleles in wild type and mrf1 mutant yeast strains produces a phenotype consistent with a reduced affinity of the mutant release factors for the ribosome, indicating that the mutations map in a release factor domain involved in ribosome binding. We also demonstrate that nonsense suppression caused by a mutation in the mitochondrial homolog of the E. coli small ribosomal protein S4 can be reversed by a slight over-expression of the MRF1 gene.     

Mutator phenotype of MUTYH-null mouse embryonic stem cells

To evaluate the antimutagenic role of a mammalian mutY homolog, namely the Mutyh gene, which encodes adenine DNA glycosylase excising adenine misincorporated opposite 8-oxoguanine in the template DNA, we generated MUTYH-null mouse embryonic stem (ES) cells. In the MUTYH-null cells carrying no adenine DNA glycosylase activity, the spontaneous mutation rate increased 2-fold in comparison with wild type cells. The expression of wild type mMUTYH or mutant mMUTYH protein with amino acid substitutions at the proliferating cell nuclear antigen binding motif restored the increased spontaneous mutation rates of the MUTYH-null ES cells to the wild type level. The expression of a mutant mMUTYH protein with an amino acid substitution (G365D) that corresponds to a germ-line mutation (G382D) found in patients with multiple colorectal adenomas could not suppress the elevated spontaneous mutation rate of the MUTYH-null ES cells. Although the recombinant mMUTYH(G365D) purified from Escherichia coli cells had a substantial level of adenine DNA glycosylase activity as did wild type MUTYH, no adenine DNA glycosylase activity was detected in the MUTYH-null ES cells expressing the mMUTYH(G365D) mutant protein. The germ-line mutation (G382D) of the human MUTYH gene is therefore likely to be responsible for the occurrence of a mutator phenotype in these patients.     

The barley amo1 locus is tightly linked to the starch synthase IIIa gene and negatively regulates expression of granule-bound starch synthetic genes

In this study of barley starch synthesis, the interaction between mutations at the sex6 locus and the amo1 locus has been characterized. Four barley genotypes, the wild type, sex6, amo1, and the amo1sex6 double mutant, were generated by backcrossing the sex6 mutation present in Himalaya292 into the amo1 'high amylose Glacier'. The wild type, amo1, and sex6 genotypes gave starch phenotypes consistent with previous studies. However, the amo1sex6 double mutant yielded an unexpected phenotype, a significant increase in starch content relative to the sex6 phenotype. Amylose content (as a percentage of starch) was not increased above the level observed for the sex6 mutation alone; however, on a per seed basis, grain from lines containing the amo1 mutation (amo1 mutants and amo1sex6 double mutants) synthesize significantly more amylose than the wild-type lines and sex6 mutants. The level of granule-bound starch synthase I (GBSSI) protein in starch granules is increased in lines containing the amo1 mutation (amo1 and amo1sex6). In the amo1 genotype, starch synthase I (SSI), SSIIa, starch branching enzyme IIa (SBEIIa), and SBEIIb also markedly increased in the starch granules. Genetic mapping studies indicate that the ssIIIa gene is tightly linked to the amo1 locus, and the SSIIIa protein from the amo1 mutant has a leucine to arginine residue substitution in a conserved domain. Zymogram analysis indicates that the amo1 phenotype is not a consequence of total loss of enzymatic activity although it remains possible that the amo1 phenotype is underpinned by a more subtle change. It is therefore proposed that amo1 may be a negative regulator of other genes of starch synthesis.     

A mutation in serca underlies motility dysfunction in accordion zebrafish

Zebrafish acquire the ability for fast swimming early in development. The motility mutant accordion (acc) undergoes exaggerated and prolonged contractions on both sides of the body, interfering with the acquisition of patterned swimming responses. Our whole cell recordings from muscle indicate that the defect is not manifested in neuromuscular transmission. However, imaging of skeletal muscle of larval acc reveals greatly prolonged calcium transients and associated contractions in response to depolarization. Positional cloning of acc identified a serca mutation as the cause of the acc phenotype. SERCA is a sarcoplasmic reticulum transmembrane protein in skeletal muscle that mediates calcium re-uptake from the myoplasm. The mutation in SERCA, a serine to phenylalanine substitution, is likely to result in compromised protein function that accounts for the observed phenotype. Indeed, direct evidence that mutant SERCA causes the motility dysfunction was provided by the finding that wild type fish injected with an antisense morpholino directed against serca, exhibited accordion-like contractions and impaired swimming. We conclude that the motility dysfunction in embryonic and larval accordion zebrafish stems directly from defective calcium transport in skeletal muscle rather than defective CNS drive.     

A dominant mutation in Escherichia coli OmpR lies within a domain which is highly conserved in a large family of bacterial regulatory proteins

Summary We have fortuitously created an in-frame insertion mutation in the cloned ompR gene of Escherichia coli in the course of an experiment involving linker insertion mutagenesis. According to the DNA sequence, the mutant protein has an insertion at the 53rd amino acid residue, which replaced the original valine, with the sequence Ala-Leu-Glu. The expression level of the mutant protein, OmpRX6, in a minicell system, is similar to that of the wild-type protein and the size of the mutant is slightly larger than the wild type by approxiately 300 daltons. This mutant was completely unable to activate porin expression as the wildtype does, and in addition, this phenotype was shown to be dominant over the wild type. Comparison of the amino acid sequence of OmpRX6 with those of a family of homologous bacterial regulatory proteins revealed that the mutation lies in a domain which is highly conserved among these proteins.     

A single amino acid substitution in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase confers resistance to the herbicide glyphosate

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19), encoded by the aroA locus, is a target site of glyphosate inhibition in bacteria. A glyphosate-resistant aroA allele has been cloned in Escherichia coli from a mutagenized strain of Salmonella typhimurium. Subcloning of this mutant aroA allele shows the gene to reside on a 1.3-kilobase segment of S. typhimurium DNA. Nucleotide sequence analysis of this mutant gene indicates a protein-coding region 427 amino acids in length. Comparison of the mutant and wild type aroA gene sequences reveals a single base pair change resulting in a Pro to Ser amino acid substitution at the 101st codon of the protein. A hybrid gene fusion between mutant and wild type aroA gene sequences was constructed. 5-Enolpyruvylshikimate-3-phosphate synthase was prepared from E. coli cells harboring this construct. The glyphosate-resistant phenotype is shown to be associated with the single amino acid substitution described above.     

Identification of signals and mechanisms of sorting of plasma membrane proteins in intestinal epithelial cells]

In epithelial cells the plasma membrane is divided into domains that are biochemically and functionally different. In intestinal cells for example the apical domain is facing the intestinal lumen and is involved in the uptake of nutriments while the basolateral domain is mediating cell-cell adhesion and signalisation. We are interested in deciphering the mechanisms underlying the creation and maintenance of such specialized domains. As an epithelial model we have used the intestinal cell line Caco-2 and we have studied the transport and sorting of the human neurotrophin receptor (p75 NTR) in these cells. Newly synthesized p75 NTR is first transported to the basolateral membrane and then is accumulated on the apical membrane after transcytosis. This final apical localization is controlled by the presence of a membrane anchor and a cluster of O-glycosylation sites located in the part of the ectodomain close to the membrane. Among the mechanisms likely to be involved in the sorting of apical components we have looked for a role of lipid-protein microdomain formation in the Golgi apparatus. These membrane microdomains are highly enriched in glycosylphosphatidyl inositol (GPI) anchored proteins, glycosphingolipids and apical proteins such as sucrase isomaltase (SI). Such a composition is also found for endocytic structures called caveolae which are made of caveolin 1. We have expressed caveolin 1 in Caco-2 cells which do not express it and also caveolin 2, a related protein of unknown function. Expression of caveolin 1 led to formation of caveolae indicating that this protein is necessary for caveolae formation while caveolin 2 is restricted to the Golgi apparatus and has no effect on caveolae formation. However Caveolin 2 increased the amount of SI incorporated in microdomains suggesting a role in recruitment into the apical pathway. The choice for a site of fusion for transport vesicles is the last step of control during exocytosis. To identify proteins involved in that step we have cloned and characterized two members of the t-SNARE family, namely syntaxin 3 and SNAP23. Syntaxin 3 is present on the apical membrane and forms a complex with SNAP23 which is also localized on the basolateral membrane where it forms a complex with syntaxin 4. Overexpression of syntaxin 3 in Caco-2 led to a decrease of SI exocytosis towards the apical membrane confirming that syntaxin 3 is involved in targeting the fusion of apical transport vesicles to the apical pole of the cells.     

The dual role of annexin II in targeting of brush border proteins and in intestinal cell polarity

Functional intestinal epithelium relies on complete polarization of enterocytes marked by the formation of microvilli and the accurate trafficking of glycoproteins to relevant membrane domains. Numerous transport pathways warrant the unique structural identity and protein/lipid composition of the brush border membrane. Annexin II (Ca(2+)-dependent lipid-binding protein) is an important component of one of the apical protein transport machineries, which involves detergent-resistant membranes and the actin cytoskeleton. Here, we investigate in intestinal Caco-2 cells the contribution of annexin II to the sorting and transport of brush border hydrolases and role in intestinal cell polarity. Downregulation of annexin II in Caco-2-A4 cell line results in a severe reduction of the levels of the brush border membrane resident enzyme sucrase isomaltase (SI) as well as structural components such as ezrin. This reduction is accompanied by a redistribution of these proteins to intracellular compartments and a striking morphological transition of Caco-2 cells to rudimentary epithelial cells that are characterized by an almost flat apical membrane with sparse and short microvilli. Concomitant with this alteration is the redistribution of the intermediate filament protein keratin 19 to the intracellular membranes in Caco-2-A4 cells. Interestingly, keratin 19 interacts with annexin II in wild type Caco-2 cells and this interaction occurs exclusively in lipid rafts. Our findings suggest a role for annexin II and K19 in differentiation and polarization of intestinal cells.     

Apical membrane proteins are transported in distinct vesicular carriers

The function of polarized epithelial cells and neurons is achieved through intracellular sorting mechanisms that recognize classes of proteins in the trans-Golgi network (TGN) and deliver them into separate vesicles for transport to the correct surface domain. Some proteins are delivered to the apical membrane after their association with membrane detergent-insoluble glycophosphatidylinositol/cholesterol (DIG) membrane microdomains [1], while some do not associate with DIGs [2-4]. However, it is not clear if this represents transport by two different pathways or if it can be explained by differences in the affinity of individual proteins for DIGs. Here, we investigate the different trafficking mechanisms of two apically sorted proteins, the DIG-associated sucrase-isomaltase (SI) and lactase-phlorizin hydrolase, which uses a DIG-independent pathway [5]. These proteins were tagged with YFP or CFP, and their trafficking in live cells was visualized using confocal laser microscopy. We demonstrate that each protein is localized to distinct subdomains in the same transport vesicle. A striking triangular pattern of concentration of the DIG-associated SI in subvesicular domains was observed. The original vesicles partition into smaller carriers containing either sucrase-isomaltase or lactase-phlorizin hydrolase, but not both, demonstrating for the first time a post-TGN segregation step and transport of apical proteins in different vesicular carriers.