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      Maintenance of Golgi structure and function depends on the integrity of ER export

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          Abstract

          The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1–COPII and Arf1–coatomer systems.

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          RADIOAUTOGRAPHIC STUDIES OF CHOLINE INCORPORATION INTO PERIPHERAL NERVE MYELIN

          This radioautographic study was designed to localize the cytological sites involved in the incorporation of a lipid precursor into the myelin and the myelin-related cell of the peripheral nervous system. Both myelinating and fully myelinated cultures of rat dorsal root ganglia were exposed to a 30-min pulse of tritiated choline and either fixed immediately or allowed 6 or 48 hr of chase incubation before fixation. After Epon embedding, light and electron microscopic radioautograms were prepared with Ilford L-4 emulsion. Analysis of the pattern of choline incorporation into myelinating cultures indicated that radioactivity appeared all along the length of the internode, without there being a preferential site of initial incorporation. Light microscopic radioautograms of cultures at varying states of maturity were compared in order to determine the relative degree of myelin labeling. This analysis indicated that the myelin-Schwann cell unit in the fully myelinated cultures incorporated choline as actively as did this unit in the myelinating cultures. Because of technical difficulties, it was not possible to determine the precise localization of the incorporated radioactivity within the compact myelin. These data are related to recent biochemical studies indicating that the mature myelin of the central nervous system does incorporate a significant amount of lipid precursor under the appropriate experimental conditions. These observations support the concept that a significant amount of myelin-related metabolic activity occurs in mature tissue; this activity is considered part of an essential and continuous process of myelin maintenance and repair.
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            COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum.

            In vitro synthesis of endoplasmic reticulum-derived transport vesicles has been reconstituted with washed membranes and three soluble proteins (Sar1p, Sec13p complex, and Sec23p complex). Vesicle formation requires GTP but can be driven by nonhydrolyzable analogs such as GMP-PNP. However, GMP-PNP vesicles fail to target and fuse with the Golgi complex whereas GTP vesicles are functional. All the cytosolic proteins required for vesicle formation are retained on GMP-PNP vesicles, while Sar1p dissociates from GTP vesicles. Thin section electron microscopy of purified preparations reveals a uniform population of 60-65 nm vesicles with a 10 nm thick electron dense coat. The subunits of this novel coat complex are molecularly distinct from the constituents of the nonclathrin coatomer involved in intra-Golgi transport. Because the overall cycle of budding driven by these two types of coats appears mechanistically similar, we propose that the coat structures be called COPI and COPII.
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              ER-to-Golgi transport visualized in living cells.

              Newly synthesized proteins that leave the endoplasmic reticulum (ER) are funnelled through the Golgi complex before being sorted for transport to their different final destinations. Traditional approaches have elucidated the biochemical requirements for such transport and have established a role for transport intermediates. New techniques for tagging proteins fluorescently have made it possible to follow the complete life history of single transport intermediates in living cells, including their formation, path and velocity en route to the Golgi complex. We have now visualized ER-to-Golgi transport using the viral glycoprotein ts045 VSVG tagged with green fluorescent protein (VSVG-GFP). Upon export from the ER, VSVG-GFP became concentrated in many differently shaped, rapidly forming pre-Golgi structures, which translocated inwards towards the Golgi complex along microtubules by using the microtubule minus-end-directed motor complex of dynein/dynactin. No loss of fluorescent material from pre-Golgi structures occurred during their translocation to the Golgi complex and they frequently stretched into tubular shapes. Together, our results indicate that these pre-Golgi carrier structures moving unidirectionally along microtubule tracks are responsible for transporting VSVG-GFP through the cytoplasm to the Golgi complex. This contrasts with the traditional focus on small vesicles as the primary vehicles for ER-to-Golgi transport.
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                Author and article information

                Journal
                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                12 November 2001
                : 155
                : 4
                : 557-570
                Affiliations
                Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, MD 20892
                Author notes

                Address correspondence to Dr. Jennifer Lippincott-Schwartz, CBMB, NICHD, NIH, Building 18T, Room 101, 18 Library Dr., Bethesda, MD 20892-5430. Tel.: (301) 402-1010. Fax: (301) 402-0078. E-mail: jlippin@ 123456helix.nih.gov

                Article
                0107045
                10.1083/jcb.200107045
                2198855
                11706049
                57241bf3-feae-4d06-a09b-73d41ce9bd63
                Copyright © 2001, The Rockefeller University Press
                History
                : 11 July 2001
                : 10 October 2001
                : 10 October 2001
                Categories
                Article

                Cell biology
                golgi apparatus; frap; gfp; copii; coatomer
                Cell biology
                golgi apparatus; frap; gfp; copii; coatomer

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