Distinct functional units of the Golgi complex in Drosophila cells

Abstract

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. Here we show that stacked fragments (units) of the Golgi complex dispersed in Drosophila imaginal disk 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 disks 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 could also be separated biochemically by immunoisolation techniques. We also show that Tn-antigen glycan is localized only in a subset of the Golgi units distributed basally in a polarized cell. We propose that the different localizations among distinct Golgi units of molecules involved in glycosylation underlie the diversity of glycan modification.fringe connection ͉ glycosylation ͉ posttranslational modification T he pattern of glycosylation is extremely diverse, yet is highly specific to each protein. How can this specificity (and diversity) be achieved? There are Ͼ300 glycosylenzymes in humans and Ͼ100 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 localization͞compartmentalization of glycosylenzymes.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 (1). Furthermore, during mitosis, the Golgi cisternae of mammalian cells become fragmented without their disassembly (2, 3). In Drosophila, Golgi cisternae are stacked but are not connected to form a ribbon at the embryonic and pupal stages even during interphase (4, 5), although there has been no evidence to date to indicate functional differences among the Golgi fragments.We previously reported a Drosophila UDP-sugar transporter, FRINGE CONNECTION (FRC), that 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 (6, 7). Interestingly, despite its broad specificity, loss-of-function studies have revealed that FRC is selectively required for Notch glycosylation, but not for GA...

Cisterna-specific Localization of Glycosylation-related Proteins to the Golgi Apparatus

“…We have previously reported that the mouse anti-120 kDa antibody stains the medial cisterna of the Golgi in Drosophila (Yano et al, 2005). In accordance with this localization, the antigen detected by the anti-120 kDa antibody is homologous to the mammalian MG-160 that is localized to the medial cisterna.…”

“…Cell biology studies revealed that the Drosophila cell has a number of dispersed small Golgi apparatuses where cis-, medialand trans-cisternae are stacked (Yano et al, 2005). This stacking of the different cisternae was shown by immunostaining using sets of cisterna-specific markers and reagents such as rabbit polyclonal antibodies against GM130 (Yano et al, 2005) and Syntaxin16 (Syx16) (Xu et al, 2002) and a mouse monoclonal antibody against a 120 kDa protein (Stanley et al, 1997) (7H6D7C2, EMD Bioscinences). The antibodies against GM130 and Syx16 stain cis-and trans-Golgi cisternae/TGN, respectively, and the anti-120 kDa antibody is used as a medial-cisternal marker.…”

An O-Glycosyltransferase Promotes Cell Adhesion during Development by Influencing Secretion of an Extracellular Matrix Integrin Ligand

“…Cell culture studies using competitive inhibitors of O-glycan extension resulted in reduced trafficking of certain proteins (33)(34)(35)(36); however, these inhibitors also influence other forms of glycosylation. Studies examining protein distribution in subregions of the Golgi complex in larval imaginal discs found that those containing O-linked GalNAc were present predominantly in basally located Golgi units, suggesting that this polarized distribution may influence apicobasal polarity in these tissues (50). Here, we provide direct evidence for the role of mucin-type O-glycans in modulating proper secretion to this basal region in vivo by examining a specific substrate in a glycosyltransferase mutant background.…”

Autophagy-Dependent Rhodopsin Degradation Prevents Retinal Degeneration inDrosophila

“…Rabbit anti-Rab7 polyclonal antibodies and rabbit anti-Arl8 polyclonal antibodies were a kind gift from Dr. Akira Nakamura (Riken CDB, Kobe, Japan) and Dr. Sean Munro (Medical Research Council-Laboratory of Molecular Biology, Cambridge, UK), respectively. Rabbit anti-GM130 antibodies were produced as previously described (Yano et al, 2005).…”