About 20 l of conditioned media was loaded per lane a

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Unformatted text preview: NUARY 2002 475 Fig. 1. DNA expression constructs used to produce rc– dragline spider silks in mammalian cells. cis Fig. 2. WesternGolgi complex of media from blot analysis medial BHK cells transfected with DNA expression trans constructs containing (A) ADF-3 and MaSpII and (B) MaSpI. About 20 l of conditioned media was loaded per lane. (A) Lane 1, ADF-3 His; lane 2, ADF-3; lane 3, ADF-33; lane 4, ADF-333; lane 5, MaSpII. (B) Lane 1, MaSpI; extracellular space lane 2, MaSpI (2). 27 www.sciencemag.org SCIENCE VOL 295 18 JANUARY 2002 remodeling of glycosyl modi cations packaging into vesicles for routing of di erent proteins to speci c nal destinations exocytosis 28 473 The compartments of the secretory pathway are topologically equivalent The Golgi is the central organelle of the secretory pathway “The soul of the cell” 29 The Nobel Prize for the secretory pathway Jim Rothman mammalian biochemistry 30 Rothman reconstituted Golgi tra cking in cell-free extracts Golgi isolated from glycosyltransferase mutant cell line! 3 ! H-GlcNAc! ! ! ! ! ! ! ! ! VSV-G! ! Mutant cells express VSV-G protein, but lack the enzyme that incorporates 3H-GlcNAc ! Randy Schekman yeast genetics 2013 Nobel Prize 31 Golgi isolated from wildtype cells! ! 3 H-GlcNAc! ! ! Medial ! ! Golgi ! ! ! ! ! ! ! cis ! ! Golgi! ! ! Medial Golgi from wild type cells can complement the glycosylation defect upon successful transport! 32 Figure 1. Intracellular Transport Pathways The scheme depicts the compartments of the secretory, lysosomal/vacuolar, and endocytic pathways. Transport steps are indicated by arrows. Colors indicate the known or presumed locations of COPII (blue), COPI (red), and clathrin (orange). Clathrin coats are heterogeneous and contain different adaptor and accessory proteins at different membranes. Only the function of COPII in ER export and of plasma membraneassociated clathrin in endocytosis are known with certainty. Less well understood are the exact functions of COPI at the ERGIC and Golgi complex and of clathrin at the TGN, early endosomes, and immature secretory granules. The pathway of transport through the Golgi stack is still being investigated but is generally believed to involve a combination of COPI-mediated vesicular transport and cisternal maturation (Pelham and Rothman, 2000). Additional coats or coat-like complexes exist but are not represented in this figure. Membrane tra c: general scheme Rothman reconstituted Golgi tra cking in cell-free extracts cargo selection vesicle budding quickly recognized vesiclecomplex. Schekman and colleagues vesicle exaggervesicle that each of these structures represented an transport secretory pathway intermediatefusion accumudocking ated that had transport carriers, (2) an enlarged ER network, or (3) a cup-shaped membranous organelle (the “Berkeley body”), which was later identified as an abnormal Golgi cytoplasm GlcNAc (-) VSV-G (+) GlcNAc (+) VSV-G (-) Figure 2. Steps of Vesicle Budding and Fusion 33 Two secretory pathways: cons...
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