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.

Fig. 1.

Stacking of cis- to transcisternal markers in Golgi fragments of Drosophila imaginal disk cells. (A-F) Colocalization of the 120-kDa protein (red in B, C, E, and F) with dGM130 (cyan in A and C) and dSYX16 (cyan in D and F) in wing disk cells, as revealed by immunostaining. (G-I) Triple staining with anti-dGM130 (blue), anti-120-kDa protein (red), and peanut agglutinin (green), revealing stacking of the three cisternal markers. Top, apical; bottom, basal. (Scale bars, 5 μm.)

Fig. 2.

Localization of FRC to Golgi units distinct from those containing SFL or RHO in imaginal disk cells. (A-C) Distinct localizations of FRC and SFL in Golgi units. Wing disks of wild-type larvae expressing MYC epitope-tagged FRC were stained with anti-MYC (red, arrows), anti-SFL (green, arrowheads), and anti-120-kDa protein (blue). The SFL signal was almost completely absent in sfl mutant embryos (data not shown), suggesting that the antibodies are specific for SFL. (D-F) Distinct localizations of FRC and RHO in Golgi units. Eye disks of wild-type larvae expressing FRC-MYC were stained with anti-MYC (red, arrows), anti-RHO (green, arrowheads), and anti-120-kDa protein. Some RHO staining did not overlap with that of the 120-kDa protein, as described (19). (Scale bars, 5 μm.)

Fig. 3.

Glycosylation state and function of SPI are not affected by frc mutation. (A and B) SPI-MYC was precipitated from homogenates of wild-type (WT) or frc^RY34 mutant larvae and subjected to SDS/PAGE followed by WGA blot analysis (A) or immunoblot analysis with anti-MYC (B). In B, homogenates were treated (+) or not (-) with PNGase F, O-glycosidase, and neuraminidase after precipitation of SPI-MYC. (C-F) Photoreceptor cells of the eye disks of third-instar larvae. Eye disks of wild-type (C) or frc^R29 (D) larvae were stained with anti-ELAV (green), and those of the wild type (E) or a frc^RY34 mutant clone, which is negatively marked with green fluorescent protein (GFP, green) and outlined by dashed lines in F, were stained with anti-BAR (red), monoclonal antibody 22C10 (blue), or anti-ELAV (green in E), as indicated. (C and D) rho and spi mutants hamper the differentiation of ELAV-positive photoreceptors, except for R8. Observation of more than one ELAV-positive photoreceptors in each ommatidium (outlined by dashed line) indicated that the function of RHO and SPI were normal. (E and F) BAR-positive R1 and R6 photoreceptors (red) were induced in the mutant clone but their locations were irregular. (G and H) Legs of the wild type (G) and a frc^R29 mutant that died in the pupal case (H). Bracts (arrows) formed normally in the frc mutant. (I and J) Wing veins of rho^ve1,frc⁺/rho^ve1,frc⁺ (I) and rho^ve1,frc^RY34/rho^ve1,frc⁺ (J) adult flies. The frc^RY34 mutation did not affect the partial loss of the distal regions of veins 3 to 5 apparent in the rho^ve1 homozygote. (Scale bars, 5 μmin C-F, 10 μmin G, and 500 μmin I.)

Fig. 4.

FRC-FNG and RHO are localized to distinct Golgi units in embryos. (A-F) FRC-MYC and RHO-HA (A and D), FRC-MYC and FNG-HA (B and E), or RHO-HA and FNG-MYC (C and F) were expressed in embryonic epidermal cells by arm-Gal4 driver. Cells were stained with anti-120kD protein (green), anti-MYC (red), and anti-HA (blue). (A and D) FRC (red) and RHO (blue) were localized to distinct Golgi units (green). (B, C, E, and F) FRC (red) and FNG (blue) were colocalized to the same Golgi units (arrows in B and E), whereas RHO (blue) and FNG (red) were localized to the distinct Golgi units (C and F). (Scale bar, 5 μm.) (G-K) Biochemical separation of FRC- and RHO-positive Golgi units. (G-I) FRC-MYC and RHO-HA (G), FRC-MYC and FNG-HA (H), or RHO-HA and FNG-MYC (I) were expressed by arm-Gal4 driver. Golgi were immunoisolated with either anti-MYC or anti-HA and subjected to SDS/PAGE followed by immunoblot analyses with anti-MYC and anti-HA. (J and K) Ratios of FRC-MYC and RHO-HA (n = 4, J), FRC-MYC and FNG-HA (n = 4, K), or RHO-HA and FNG-MYC (n = 4, K) in each immunoisolate are shown.

Fig. 5.

Synthesis of Tn antigen is restricted to a subset of Golgi units. (A-C) The undifferentiated cells of eye disks before progression of the morphogenetic furrow are shown in vertical view (A: top, apical; bottom, basal) and in horizontal views (B, apical side; C, basal side). Immunostaining revealed that Tn antigen (cyan) was mostly restricted to the basally located Golgi units stained by anti-dGM130 (red). Arrows indicate Tn antigen localized in the Golgi units. (Scale bars, 5 μm.) (D) Percentages of Tn antigen-positive (cyan) and Tn antigen-negative (red) Golgi units at various horizontal levels of imaginal disk cells. Numbers of the Golgi units counted at each surface are 301 (0.0-μm depth), 221 (1.5 μm), 158 (3.0 μm), 233 (4.5 μm), 156 (6.0 μm), 152 (7.5 μm), and 120 (9.0 μm).

Fig. 6.

Colocalization of FRC and SFL in Drosophila embryos and schematic models of the Golgi complex in Drosophila and mammalian cells. (A-C) A Drosophila embryo expressing FRC-MYC at stage 14 immunostained with anti-MYC (red), anti-120-kDa protein (blue), and anti-SFL (green). Arrows indicate colocalization of FRC and SFL. (Scale bar, 5 μm.) (D) Distinct Golgi units are dispersed in Drosophila cells (Left) but are connected in mammalian cells (Right). Different units contain different sets of molecules involved in posttranslational modifications such as glycosylation and proteolytic processing. Secretory or membrane proteins such as NOTCH, SPI, and GAG core proteins may be transported to appropriate Golgi units in Drosophila cells.