A Cytosolic Complex of p 62 and rab 6 Associates with TGN 38 / 41 and Is Involved in Budding of Exocytic Vesicles from the trans-Golgi Network

TGN38/41, an integral membrane protein predominantly localized to the trans-Golgi network, has been shown to cycle to the plasma membrane and return to the TGN within 30 min. (Ladinsky, M. S., and K. E. Howell. 1992. Eur. J. Cell BioL 59:92-105). In characterizing the proteins which associate with TGN38/41, a peripheral 62-kD protein, two forms of rab6 and two other small GTP-binding proteins were identified by coimmunoprecipitation. However, •90% of the 62-kD protein is cytosolic and is associated with the same subset of small GTP-binding proteins. Both the membrane and cytoplasmic complexes were characterized by sizing column fractionation and velocity sedimentation. The membrane complex was ,',,250 kD (11.6 S) consisting of the cytosolic complex and a heterodimer of TGN38/41 (160 kD). The cytosolic complex was '~86 kD (6.1 S) consisting of p62 and one small GTP-binding protein. Preliminary evidence indicates that phosphorylation of the p62 molecule regulates the dissociation of the cytosolic complex from TGN38/41. Functionally the cytosolic p62 complex must bind to TGN38/41 for the budding of exocytic transport vesicles from the TGN as assayed in a cell-free system (Salamero, J., E. S. Sztul, and K. E. Howell. 1990. Proc. Natl. Acad. Sci. USA. 87:7717-7721). Interference with p62, rab6 or TGN38, and TGN41 cytoplasmic domains by immunodepletion or competing peptides completely inhibited the budding of exocytic transport vesicles. These results support an essential role for interaction of the cytosolic p62/rab6 complex with TGN38/41 in budding of exocytic vesicles from the TGN. T HE molecular mechanism of vesicular transport, by which both proteins and lipids move from one subcellular compartment to another, has been a topic of intense investigation recently (for reviews see Rothman and Orci, 1992; Pryer et al., 1992; Mellman and Simons, 1992). Cell-free assays combined with genetic studies in Saccharomyces cerevisiae have allowed the identification of numerous molecules involved in the vesicular transport process. Many cell-free assays measure the entire process, departure of a marker from one compartment and arrival at the next (for reviews see Melan~on et al., 1991; Sztul et al., 1992). However, vesicular transport can be divided into many individual processes and cell-free assays can be designed to study each individual step. Additional impetus to the vesicular transport field has come from the consistent finding that transport between each donor and acceptor compartment appears to be regulated by distinct members of the rab subfamily of the ras-related low molecular weight GTPbinding proteins (for reviews see Balch, 1990; Gruenberg and Clague, 1992; Pfeffer, 1992). Please address all correspondence to K. E. Howell, Department of Cellular and Structural Biology, University of Colorado School of Medicine, Denver, CO 80262. Dr. J. Salamero's current address is Centre d'Immunologie-MarseflleLuminy, Case 906, 13288 MarseiUe, Cedex 9, France. S. M. Jones and J. R. Crosby contributed equally to the work in this paper. We have been studying the budding of vesicles from an immobilized stacked Golgi fraction and have found that formation of exocytic transport vesicles from the TGN is both rapid and efficient (Salamero et al., 1990). To study the budding from the TGN in more detail, we have focused on a type 1, integral membrane protein of the TGN, TGN38; the cDNA was isolated, sequenced, and expressed by Luzio et al. (1990). Recently, an isoform of the TGN38 eDNA was identified, TGN41, which has an 8 base change, including a 5-bp insertion resulting in an extended cytoplasmic domain of 23 amino acids (Reaves et al., 1992). Most studies on TGN38 have used antibodies which recognize the luminal domain, as shown by epitope mapping of different polyclonal antibodies (Wilde et al., 1992). Since the luminal domain is identical in both isoforms, experiments to date have studied TGN38/41 (Luzio et al., 1990; Robinson, 1990; Hunziker et al., 1991; Lippincott-Schwartz et al., 1991; Wood et al., 1991; Ladinsky and Howell, 1992; Reaves and Bantings, 1992; Reaves et al., 1993; Humphrey et al., 1993; Bos et al., 1993). Signals in the cytoplasmic domain of TGN38141 have been shown to be responsible for its predominant TGN localization (Luzio et al., 1990; Humphrey et al., 1993; Bos et al., 1993). Although the predominant localization is the TGN, the molecule rapidly cycles to the PM and returns to its resident compartment within 30 min (Ladinsky and Howell, © The Rockefeller University Press, 0021-9525/93/08/775/14 $2.00 The Journal of Cell Biology, Volume 122, Number 4, August 1993 775-788 775 on N ovem er 9, 2017 jcb.rress.org D ow nladed fom 1992; Reaves et al., 1993). These findings led us to investigate the proteins that associate with TGN38/41 to identify molecules involved in the trafficking process. To amplify our signal, we started with a stacked Golgi fraction isolated from rat liver. The secretory proteins were removed by carbonate washing and the membranes were solubilized in CHAPS to form mixed detergent/lipid micelles. Using this preparation, we have identified a protein at 62 kD and a select subset of small GTP-binding proteins, including rab6 which coimmunoprecipitate with TGN38/41. The association of these proteins also has been demonstrated by two other methods, sizing column chromatography and velocity sedimentation in sucrose gradients. Rab6 has previously been localized to the Golgi complex and peripheral Golgi vesicles (Goud et al., 1990; Yang et al., 1992; Antony et al., 1992). Approximately 90% of p62 is cytosolic and is associated with four small GTP-binding proteins. The 10% of p62 which is bound to the Golgi membrane is in a complex with TGN38/41 and all of the GTP-binding proteins which are found associated with the cytosolic complex. Phosphorylation may regulate the dissociation of the 1362 cytosolic complex from TGN38/41, and hence the membrane. Since monomeric GTP-binding proteins are thought to function by catalyzing the assembly of interacting molecules (for review see Bourne et al., 1990; Sztul et al., 1992), the TGN38/4 l/GTP-binding protein complex is an excellent candidate for involvement in the vesicular transport process. Functional studies using a cell-free budding assay reveals that the p62 cytosolic complex must associate with the cytoplasmic domain of TGN38/41 for the formation of exocytic transport vesicles from the TGN. Materials and Methods