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Zygotically transcribed genes

Genes Regulating Golgi Apparatus

Distinct functional units of the Golgi complex in Drosophila cells

Genes involved in Golgi apparatus function

Protein processed by the Golgi apparatus

Distinct functional units of the Golgi complex in Drosophila cells

A striking variety of glycosylation occur in the Golgi complex in a protein-specific manner, but how this diversity and specificity are achieved remains unclear. This study shows that stacked fragments (units) of the Golgi complex dispersed in Drosophila imaginal disc cells are functionally diverse. The UDP-sugar transporter Fringe-Connection (Frc) is localized to a subset of the Golgi units distinct from those harboring Sulfateless (Sfl), which modifies glucosaminoglycans (GAGs), and from those harboring the protease Rhomboid (Rho), which processes the glycoprotein Spitz (Spi). Whereas the glycosylation and function of Notch are affected in imaginal discs of frc mutants, those of Spi and of GAG core proteins are not, even though Frc transports a broad range of glycosylation substrates, suggesting that Golgi units containing Frc and those containing Sfl or Rho are functionally separable. Distinct Golgi units containing Frc and Rho in embryos can also be separated biochemically by immunoisolation techniques. Tn-antigen glycan is shown to be localized only in a subset of the Golgi units distributed basally in a polarized cell. It is proposed that the different localizations among distinct Golgi units of molecules involved in glycosylation underlie the diversity of glycan modification (Yano, 2005).

The pattern of glycosylation is extremely diverse, yet is highly specific to each protein. How can this specificity (and diversity) be achieved? There are >300 glycosylenzymes in humans and >100 in Drosophila, but is their enzymatic specificity sufficient to explain the precise modification of all substrates? One possible mechanism that might also contribute to the specific (and diverse) pattern of glycosylation would be the localization/compartmentalization of glycosylenzymes (Yano, 2005).

The Golgi complex, where protein glycosylation takes place, has been regarded as a single functional unit, consisting of cis-, medial-, and transcisternae in mammalian cells. However, the three-dimensional reconstruction of electron microscopic images of the mammalian Golgi structure has suggested the existence of more than one Golgi stack, with the individual stacks being connected into a ribbon by tubules bridging equivalent cisternae. Furthermore, during mitosis, the Golgi cisternae of mammalian cells become fragmented without their disassembly. In Drosophila, Golgi cisternae are stacked but are not connected to form a ribbon at the embryonic and pupal stages even during interphase, although there has been no evidence to date to indicate functional differences among the Golgi fragments (Yano, 2005).

A Drosophila UDP-sugar transporter, Fringe connection (Frc) transports a broad range of UDP-sugars that can be used for the synthesis of various glycans, including N-linked types, GAGs, and mucin types. Interestingly, despite its broad specificity, loss-of-function studies have revealed that Frc is selectively required for Notch glycosylation, but not for GAG synthesis. This observation prompted a study at Frc localization; in this study, it was found that Frc is localized only to a subset of Golgi fragments in Drosophila discs and embryos (Yano, 2005).

Frc, Sfl, a glycosylenzyme of GAGs, and Rho, a processing enzyme of Spi glycoprotein, are localized to distinct Golgi fragments, which are referred to as 'Golgi units,' in Drosophila cells. frc mutants do not exhibit defects in the glycosylation and function of Spi nor do they exhibit defects in glycosylation or function of GAG core proteins. Moreover, biochemically separated distinct Golgi units containing Frc and Rho were isolated by immunoisolation technique. This study clearly shows that there are functionally distinct Golgi units in a Drosophila cell (Yano, 2005).

The Golgi complex is a stack of cis-, medial-, and transcisternae in mammalian cells. In contrast, Golgi markers often do not overlap with each other in Saccharomyces cerevisiae, in which the Golgi cisternae are not stacked but disassembled. The Golgi cisternae of Drosophila are stacked but are not connected to form a ribbon at the embryonic and pupal stages even during interphase. To determine whether Drosophila imaginal disc cells have assembled or disassembled Golgi cisternae, the localizations were compared of the cis-cisternal marker dGM130, the transcisternal marker dSyntaxin16 (dSyx16), and the Golgi-tethered 120-kDa protein, which is commonly used to detect the Golgi complex in Drosophila. The 120-kDa protein was identified by immunoaffinity purification and protein sequencing as a Drosophila homolog of the vertebrate 160-kDa medial Golgi sialoglycoprotein (MG160), which resides uniformly in the medial-cisternae of the Golgi apparatus in vertebrate cells. An antibody specific for the 120-kDa protein also stained numerous Golgi fragments in imaginal disc cells. More than 80% of immunoreactivity for the 120-kDa protein colocalizes with both dGM130 and dSYX16, suggesting that 120-kDa protein-positive fragments of the Golgi complex indeed comprise assembled cisternae; these fragments will be referred to as 'Golgi units'. The distributions of the 120-kDa protein, dGM130, and peanut agglutinin (PNA), another transcisternal marker, also shows that the markers are closely apposed but not identical, suggesting that the Golgi units are polarized. Interestingly, most of the PNA-positive transcisternae are oriented toward the basal side of the cell, within the Golgi complex, whereas most of the GM130-positive cis-cisternae are oriented toward the apical side of the cell. The cis-to-trans polarity of each Golgi unit thus appears to be correlated with the apico-basal polarity of the disc cells (Yano, 2005).

Drosophila mutant larvae defective in the UDP-sugar transporter Frc manifest a highly selective phenotype: the lack of Notch glycosylation in the presence of normal GAG synthesis (Goto, 2001). This limited phenotype was unexpected, given that Frc exhibits a broad specificity for UDP sugars used in the synthesis of various glycans including N-linked types, GAGs, and mucin types. However, given that the frcR29 allele studied previously (Goto, 2001) is hypomorphic, whether the selective glycosylation defect might be a consequence of partial loss of Frc activity was examined. With the use of imprecise excision, a new allele, frcRY34, was generated the presence of which results in the death of most larvae during the second-instar stage, much earlier than the death induced by frcR29. Real-time PCR analysis revealed that the amount of frc transcripts in the second-instar larvae of frcRY34 or frcR29 mutants was 4.2% and 24.4% of that in the wild type, respectively. About 1 kb of the gene, including the transcription initiation site, was deleted in the frcRY34 allele. Together, these observations suggest that frcRY34 is essentially a null allele (Yano, 2005).

Clonal cells of the frcRY34 mutant exhibit normal levels of GAGs, as detected by immunostaining with the 3G10 antibody, whereas the amount of GAGs was reduced in clones of tout-velu (ttv) mutant cells. Given that GAGs are required for signaling by Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp),the expression was examined of corresponding target genes [patched (ptc) for Hh signaling and Dll for Wg and Dpp signaling] in the wing discs of the frcRY34 mutant. Expression of ptc and that of Dll in the ventral compartment of the wing discs were unaffected in the mutant clones, suggestive of normal GAG function (Yano, 2005).

Given that Notch glycosylation by Fringe (Fng), a fucose-specific ß1,3-N-acetylglucosaminyltransferase, requires Frc activity, Notch glycosylation was examined in the frcRY34 mutant. The frcRY34 mutant clones in the dorsal compartment, but not those in the ventral compartment, of the wing discs induce wg expression at their borders, as has been observed with fng mutant clones, suggesting that Notch glycosylation is impaired in the frcRY34 mutant. The ectopic expression of Wg induced by the frcRY34 mutant clones is likely responsible for the observed induction of Dll expression in the dorsal compartment (Yano, 2005).

To determine why the loss of a UDP-sugar transporter with a broad specificity selectively affects Notch glycosylation, the subcellular localization of Frc was examined. Frc tagged with the Myc epitope was expressed in imaginal discs under the control of the arm-Gal4 driver. The Gal4-induced expression of Frc-Myc rescues the frc mutant phenotype, suggesting that Frc-Myc is functional and properly localized. Immunostaining of imaginal discs of wild-type larvae expressing Frc-Myc with antibodies to Myc and to the 120-kDa protein revealed that Frc localizes to only a small subset of Golgi units. Thus, it is hypothesized that the Golgi units might be functionally heterogeneous, and that those containing Frc might modify some proteins, including Notch, but not others (Yano, 2005).

To test this hypothesis, the localizations of various molecules involved in protein modification in the Golgi complex were compared with that of Frc. It was found that Sfl is also restricted to a subset of Golgi units, but that its distribution does not overlap with that of Frc. This differential localization of Sfl and Frc might thus explain the observation that frc mutant clones in wing discs do not show any defect in GAG synthesis by Sfl (Yano, 2005).

The Spi-processing enzyme Rho was also localized to a subset of Golgi units distinct from those containing Frc, in addition to its presence in other compartments. This result indicates the existence of at least two types of Golgi units, those containing Rho and those containing Frc. To determine whether these two types of Golgi units differ functionally, the glycosylation state and function of Spi were examined in frc mutants (Yano, 2005).

Given that the extent of Notch glycosylation, as detected by wheat germ agglutinin (WGA), is markedly reduced in frc mutants compared with that in the wild-type background (Goto, 2001), whether the WGA-reactive glycan of Spi is also affected by frc mutation was examined. Myc epitope-tagged Spi was expressed in the wild type or the frcRY34 mutant. Spi-Myc was then precipitated from larval homogenates with antibodies to Myc and was examined for its glycosylation by SDS/PAGE and subsequent blot analysis with WGA. The reactivity of the Spi glycan with WGA was similar in the frc mutant and in the wild type. Whether the frcRY34 mutation affects the Spi glycan was examined by mobility shift analysis. The electrophoretic mobility of glycosylated Spi from the wild type was also similar to that from the frc mutant. Deglycosylation of Spi by neuraminidase, peptide-N-glycosidase (PNGase) F, and O-glycanases also increased its mobility to the same extent in wild-type and frc mutant larvae, suggesting that the core protein is not affected by the frc mutation. Together, these results indicate that the function of Frc is not necessary for formation of the Spi glycan (Yano, 2005).

Spi function was evaluated by examining developmental processes such as photoreceptor recruitment and bract formation, both of which require Spi activation. During eye development, although Spi is not necessary for the primary induction of the photoreceptor R8, it is required for the subsequent recruitment of R1 to R7. Given that photoreceptors R1 to R8 express ELAV and that R1 and R6 express Bar, the expression of these proteins was examined in frc mutants. In mutants harboring the hypomorphic allele frcR29, all photoreceptors are normally induced, although their direction is irregular as seen in fringe or Notch mutants. Similar results were obtained by clonal analysis of frcRY34 mutants. Spi function in photoreceptor recruitment thus did not appear to be impaired in the frc mutants. The frcR29 mutant also formed normal bracts on malformed legs. Tests were performed for genetic interaction between rho and frc mutations in wing vein formation. The rhove1 mutant is viable but shows partial loss of L3-5 veins. This phenotype is also apparent in rhove1, frcRY34/rhove1, frc+ flies, suggesting that Frc does not affect Rho function. From these results, it is concluded that the function of the Rho-Spi pathway is not affected by frc mutation (Yano, 2005).

To confirm that the Golgi units containing Frc and those containing Rho are distinct, whether these Golgi units could be selectively isolated was tested by using antibodies to Myc (for Myc-tagged Frc) or HA (for HA-tagged Rho). Because it is difficult to collect enough of the imaginal discs, the starting material was switched to embryos, and whether Frc and Rho are also localized to distinct Golgi units in embryos was examined. Frc-Myc and Rho-HA were coexpressed in the embryos by the arm-Gal4 driver; immunostaining with antibodies to Myc and to HA revealed that the Golgi units containing Frc-Myc (45.4% of total Golgi units) and those containing Rho-HA (43.0% of total Golgi units) are largely distinct: only 11.6% of total Golgi units were positive for both Frc-Myc and Rho-HA. Immunoisolation was attempted from embryonic lysates by using either antibody to Myc or HA and how much Frc-Myc and Rho-HA were coisolated in each immunoisolate was examined. When Frc-Myc was immunoisolated with an antibody to Myc, the recovery of Frc-Myc was 5.7 times greater than that of Rho-HA. Moreover, when Rho-HA was immunoisolated with an antibody to HA, the recovery of Rho-HA was 18.3 times greater than that of Frc-Myc. The immunoblot analysis of these immunoisolates with the anti-120-kDa antibody confirmed that the Golgi units were concentrated in these immunoisolates. These results support the notion that Frc-Myc-containing fraction is distinct and can be separated from Rho-HA-containing fraction (Yano, 2005).

Whether these distinct Golgi units contain different constituents was examined. Fringe (Fng) is one of the candidate molecules that may be colocalized with Frc. Therefore, expression of ectopically expressed Fng was examined in Rho- and Frc-containing immunoisolates. It was found that expression of Fng in Frc-containing immunoisolates was 26 times greater than in Rho-containing immunoisolates, supporting the idea that Fng is localized in the Frc-positive Golgi units rather than the Rho-positive Golgi units. It was also confirmed by immunostaining analysis that Fng colocalizes mostly with Frc (88.1% of the FNG-positive Golgi units), but not with Rho (16.6% of the Fng-positive Golgi units), by immunostaining analysis (Yano, 2005).

The data suggest that different Golgi units perform different functions, a notion that is also supported by the observation that Tn antigen (O-linked N-acetylgalactosamine) was detected in only a subset of Golgi units in imaginal eye disc cells. In addition, it was found that most of these Tn antigen-positive Golgi units are distributed in the basal region of the disc cells, suggesting that the differential distribution of Golgi units might contribute to the apicobasal polarity of glycan distribution (Yano, 2005).

In contrast to the larval stage, Frc is required for GAG synthesis at the early embryonic stage (Goto, 2001; Selva, 2001). To determine why the Frc requirement for GAG synthesis differs between the embryonic and larval stages, embryos expressing Frc-Myc were stained with antibodies to Sfl and to Myc. Sfl was found to be colocalized with Frc, likely explaining the importance of Frc for GAG synthesis at the embryonic stage. In addition, this embryonic requirement of Frc for GAG synthesis excludes the possibility that the selective defects in Notch and not in GAG synthesis observed in frc mutant larvae are caused by the selective Frc-dependent transport of a subset of UDP-sugars used only for glycosylation of Notch but not for GAGs synthesis (Yano, 2005).

It summary, these results provide evidence for the existence of functionally distinct Golgi units in Drosophila cells. Such functional heterogeneity of Golgi units is likely responsible for the diversity of protein glycosylation. At least two types of Golgi units containing either Frc or Sfl are present in larval disc cells. Two distinct sets of proteins, exemplified by Notch and GAG core proteins, might thus be selectively transported to Frc- or Sfl-containing Golgi units, respectively, where they undergo glycosylation by different sets of molecules (Yano, 2005).

The variety of Golgi units might be established by separate transport of secretory proteins and glycosylenzymes from the endoplasmic reticulum (ER) to the distinct Golgi units. In yeast, glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in vesicles distinct from those containing other secretory protein. Given that the GAG core protein Dally in Drosophila is anchored to the membrane by GPI, it is possible that Dally and Notch are loaded into distinct vesicles as they exit the ER (Yano, 2005).

Combinations of glycosylenzymes and transporters, such as Sfl and Frc, contained in Golgi units of Drosophila differ not only between embryos and larval disc cells but also among cell types. For example, Frc is localized to all Golgi units in salivary gland cells at the larval stage. It has also been shown that all of the Golgi complexes dispersed in oocytes may have the ability to process the Gurken precursor protein, which is usually cleaved in a subset of the Golgi complexes residing in the dorso-anterior region. The Golgi units may thus be altered in a manner dependent on development, cell type, and signaling processes (Yano, 2005).

The functional diversity of Golgi units also might contribute to the polarized distribution of glycans along the apicobasal axis of cells. It was found that Tn antigen is synthesized in the basal Golgi units of larval disc cells. Furthermore, certain types of glycans are distributed along the apicobasal axis of pupal ommatidia. These glycans might thus be synthesized differentially in the Golgi units that are asymmetrically distributed along the apicobasal axis and then be secreted at either the apical or basal cell surface (Yano, 2005).

Whereas Golgi units are dispersed throughout Drosophila cells, the Golgi complex in mammalian cells is thought to be a single entity that is located in the pericentriolar region through its association with the microtubule-organizing center in interphase and which is fragmented at the onset of mitosis. The Golgi fragments apparent in mammalian cells during mitosis are highly similar to the Golgi units of Drosophila cells in both electron and confocal microscopic images. The mammalian Golgi complex during interphase may therefore be comprised of functionally distinct units that are associated with the microtubule-organizing center and connected with each other (Yano, 2005).


Goto, S., Taniguchi, M., Muraoka, M., Toyoda, H., Sado, Y., Kawakita, M. and Hayashi, S. (2001). UDP-sugar transporter implicated in glycosylation and processing of Notch. Nat. Cell Biol. 3: 816-822. 11533661

Selva, E. M., Hong, K., Baeg, G. H., Beverley, S. M., Turco, S. J., Perrimon, N. and Hacker, U. (2001). Dual role of the fringe connection gene in both heparan sulphate and fringe-dependent signalling events. Nat. Cell Biol. 3: 809-815. 11533660

Yano, H., et al. (2005). Distinct functional units of the Golgi complex in Drosophila cells. Proc. Natl. Acad. Sci 102: 13467-13472. 16174741

Zygotically transcribed genes

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