A cytosolic complex of p62 and rab6 associates with TGN38/41 and is involved in budding of exocytic vesicles from the trans-Golgi network

Abstract

Abstract. 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.

The TGN38 glycoprotein contains two non-overlapping signals that mediate localization to the trans-Golgi network.

“…TGN38 was originally identified as a resident of the TGN (Luzio et al, 1990) but more recent work has shown that it cycles between the TGN and the cell surface (Jones et al, 1993;Reaves et al, 1993). Movement to the cell surface occurs via exocytic vesicles (Jones et al, 1993) and is followed by retrieval which requires a signal present in the cytoplasmic domain that has been localized to the sequence SDYQRL (Bos et al, 1993;Humphrey et ai., 1993;Wong and Hong, 1993).…”

“…Movement to the cell surface occurs via exocytic vesicles (Jones et al, 1993) and is followed by retrieval which requires a signal present in the cytoplasmic domain that has been localized to the sequence SDYQRL (Bos et al, 1993;Humphrey et ai., 1993;Wong and Hong, 1993). Retrieved TGN38 first moves to the endosomes and then to the TGN (Bos et al, 1993;Humphrey et al, 1993).…”

Differential distribution of alpha subunits and beta gamma subunits of heterotrimeric G proteins on Golgi membranes of the exocrine pancreas.

“…6) with A R F and with the trans-Golgi markers, TGN38 (25), rab6 (3,20), and caveolin (16). A R F (Fig.…”

Characterization of the Golgi Complex Cleared of Proteins in Transit and Examination of Calcium Uptake Activities

“…We initiated this project in studying the Golgi cleared of proteins in transit with the following long-range objectives: to characterize the basic structure of the Golgi ribbon and to obtain a Golgi fraction where in vivo structure remained relatively intact in order to identify endogenous Golgi proteins and activities. This latter objective led us to revise the Golgi fractionation procedure we had been using for our functional assays of vesicle budding from the trans-Golgi network (TGN; Salamero et al, 1990;Jones et al, 1993). The modified procedure provides highly enriched intact stacked Golgi fractions with and without proteins in transit.…”