The budding yeast Pichia pastoris contains ordered Golgi stacks next to discrete transitional endoplasmic reticulum (tER) sites, making this organism ideal for structure-function studies of the secretory pathway. Here, we have used P. pastoris to test various models for Golgi trafficking.The experimental approach was to analyze P. pastoris tER-Golgi units by using cryofixed and freeze-substituted cells for electron microscope tomography, immunoelectron microscopy, and serial thin section analysis of entire cells. We find that tER sites and the adjacent Golgi stacks are enclosed in a ribosome-excluding "matrix." Each stack contains three to four cisternae, which can be classified as cis, medial, trans, or trans-Golgi network (TGN). No membrane continuities between compartments were detected. This work provides three major new insights. First, two types of transport vesicles accumulate at the tER-Golgi interface. Morphological analysis indicates that the center of the tER-Golgi interface contains COPII vesicles, whereas the periphery contains COPI vesicles. Second, fenestrae are absent from cis cisternae, but are present in medial through TGN cisternae. The number and distribution of the fenestrae suggest that they form at the edges of the medial cisternae and then migrate inward. Third, intact TGN cisternae apparently peel off from the Golgi stacks and persist for some time in the cytosol, and these "free-floating" TGN cisternae produce clathrin-coated vesicles. These observations are most readily explained by assuming that Golgi cisternae form at the cis face of the stack, progressively mature, and ultimately dissociate from the trans face of the stack. INTRODUCTIONThe Golgi apparatus consists of flattened membrane cisternae that are usually organized into stacks (Berger and Roth, 1997). Newly synthesized biosynthetic cargo molecules exit the endoplasmic reticulum (ER) in COPII-coated vesicles and enter the cis cisterna of the Golgi (Farquhar and Hauri, 1997;Barlowe, 2002). These cargo molecules then occur in medial and trans-cisternae. In the trans-Golgi network (TGN), the cargo molecules are sorted into different types of carriers for delivery to the plasma membrane and other destinations (Mellman and Simons, 1992). Although this basic scheme is well established, the pathway of intra-Golgi transport is still being investigated. Anterograde Golgi transport has been proposed to occur via vesicular intermediates, membrane continuities, cisternal maturation, or a combination of these mechanisms (Beznoussenko and Mironov, 2002). The extent to which a given mechanism operates may vary with the cell type and the stage of the cell cycle (Pelham and Rothman, 2000;Marsh and Howell, 2002). Considerable evidence now favors cisternal maturation as a major route for intra-Golgi transport (Pelham, 2001;Storrie and Nilsson, 2002), but the generality and several key predictions of this model remain to be verified.We are using electron microscopy to test predictions of the different Golgi-trafficking models. Because some of the...

3D electron tomography studies of the structure of the mammalian Golgi complex have led to four functional predictions (1). The sorting and exit site from the Golgi comprises two or three distinct trans‐cisternae (2). The docking of vesicular–tubular clusters at the cis‐face and the fragmentation of trans‐cisternae are coordinated (3). The mechanisms of transport through, and exit from, the Golgi vary with physiological state, and in different cells and tissues (4). Specialized trans‐ER functions in the delivery of ceramide to sphingomyelin synthase in the trans‐Golgi membrane, for the regulated sorting via sphingolipid‐cholesterol‐rich domains. These structure‐based predictions can now be tested using a variety of powerful cell and molecular tools.

The Golgi complex functions to posttranslationally modify newly synthesized proteins and lipids and to sort them to their sites of function. In this study, a stacked Golgi fraction was isolated by classical cell fractionation, and the protein complement (the Golgi proteome) was characterized using multidimensional protein identification technology. Many of the proteins identified are known residents of the Golgi, and 64% of these are predicted transmembrane proteins. Proteins localized to other organelles also were identified, strengthening reports of functional interfacing between the Golgi and the endoplasmic reticulum and cytoskeleton. Importantly, 41 proteins of unknown function were identified. Two were selected for further analysis, and Golgi localization was confirmed. One of these, a putative methyltransferase, was shown to be arginine dimethylated, and upon further proteomic analysis, arginine dimethylation was identified on 18 total proteins in the Golgi proteome. This survey illustrates the utility of proteomics in the discovery of novel organellar functions and resulted in 1) a protein profile of an enriched Golgi fraction; 2) identification of 41 previously uncharacterized proteins, two with confirmed Golgi localization; 3) the identification of arginine dimethylated residues in Golgi proteins; and 4) a confirmation of methyltransferase activity within the Golgi fraction.

The interaction between mannan polysaccharides and cellulose microfibrils contributes to cell wall properties in some vascular plants, but the molecular arrangement of mannan in the cell wall and the nature of the molecular bonding between mannan and cellulose remain unknown. Previous studies have shown that mannan is important in maintaining Arabidopsis () seed mucilage architecture, and that Cellulose Synthase-Like A2 (CSLA2) synthesizes a glucomannan backbone, which Mannan α-Galactosyl Transferase1 (MAGT1/GlycosylTransferase-Like6/Mucilage Related10) might decorate with single α-Gal branches. Here, we investigated the ratio and sequence of Man and Glc and the arrangement of Gal residues in Arabidopsis mucilage mannan using enzyme sequential digestion, carbohydrate gel electrophoresis, and mass spectrometry. We found that seed mucilage galactoglucomannan has a backbone consisting of the repeating disaccharide [4)-β-Glc-(1,4)-β-Man-(1,], and most of the Man residues in the backbone are substituted by single α-1,6-Gal. CSLA2 is responsible for the synthesis of this patterned glucomannan backbone and MAGT1 catalyses the addition of α-Gal. In vitro activity assays revealed that MAGT1 transferred α-Gal from UDP-Gal only to Man residues within the CSLA2 patterned glucomannan backbone acceptor. These results indicate that CSLAs and galactosyltransferases are able to make precisely defined galactoglucomannan structures. Molecular dynamics simulations suggested this patterned galactoglucomannan is able to bind stably to some hydrophilic faces and to hydrophobic faces of cellulose microfibrils. A specialization of the biosynthetic machinery to make galactoglucomannan with a patterned structure may therefore regulate the mode of binding of this hemicellulose to cellulose fibrils.