Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38).
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ABSTRACT:
SUBMITTER: Luzio JP
PROVIDER: S-EPMC1131683 | biostudies-other | 1990 Aug
REPOSITORIES: biostudies-other
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Project description:TGN38 is an integral membrane protein previously shown to be predominantly localized to the trans-Golgi network (TGN) of cells by virtue of a signal contained within its cytoplasmic 'tail' [Luzio, Brake, Banting, Howell, Braghetta & Stanley (1990) Biochem. J. 270, 97-102]. We now (i) describe the isolation of cDNA clones encoding an isoform of TGN38, (ii) present the sequence of that isoform and (iii) describe the production and use of antibodies which specifically recognize the new isoform. This isoform, designated TGN41, is also predominantly localized to the TGN. The only sequence differences between the protein coding regions of cDNA clones encoding TGN38 and those encoding TGN41 occur within the region specifying the cytoplasmic tails of the two proteins. The TGN localization signal is shown to be within the sequence common to both proteins.
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Project description:Observations of Golgi fragmentation upon introduction of G protein ?? (G??) subunits into cells have implicated G?? in a pathway controlling the fission at the trans-Golgi network (TGN) of plasma membrane (PM)-destined transport carriers. However, the subcellular location where G?? acts to provoke Golgi fragmentation is not known. Additionally, a role for G?? in regulating TGN-to-PM transport has not been demonstrated. Here we report that constitutive or inducible targeting of G?? to the Golgi, but not other subcellular locations, causes phospholipase C- and protein kinase D-dependent vesiculation of the Golgi in HeLa cells; Golgi-targeted ?(1)?(2) also activates protein kinase D. Moreover, the novel G?? inhibitor, gallein, and the G??-sequestering protein, GRK2ct, reveal that G?? is required for the constitutive PM transport of two model cargo proteins, VSV-G and ss-HRP. Importantly, Golgi-targeted GRK2ct, but not a PM-targeted GRK2ct, also blocks protein transport to the PM. To further support a role for Golgi-localized G??, endogenous G? was detected at the Golgi in HeLa cells. These results are the first to establish a role for Golgi-localized G?? in regulating protein transport from the TGN to the cell surface.
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Project description:Okadaic acid (OA) is a protein phosphatase inhibitor which has, among other properties, previously been shown to induce a fragmentation of the cisternae of the Golgi stack [for review, see Lucocq (1992) J. Cell Sci. 103, 875-880]. The effects of OA an reversible and mimic intracellular events which occur during mitosis. To date, due to a lack of endogenous marker proteins, the effects of OA on the trans-Golgi network (TGN) has not been studied. Certain drugs, e.g. Brefeldin A (BFA), have different effects on the morphology of the Golgi stack and the TGN; it is therefore relevant to ask what effect(s) OA has on the TGN. We now present data from a study in which we have used antibodies to TGN38, an integral membrane protein predominantly localized to the TGN of rat NRK cells [Luzio, Brake, Banting, Howell, Braghetta and Stanley (1990) Biochem. J. 270, 97-102], to investigate the effects of OA on this organelle. OA induces a reversible fragmentation of the TGN. This fragmentation occurs with similar kinetics to that observed within the Golgi stack, and is independent of protein synthesis. The sensitivity of the TGN to OA is similar to that of the Golgi stack. The fragmentation of the TGN induced by OA also leads to a 10-fold increase in the level of TGN38 expressed at the plasma membrane.
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