Sequence and overexpression of GPP130/GIMPc: evidence for saturable pH-sensitive targeting of a type II early Golgi membrane protein.

Abstract

It is thought that residents of the Golgi stack are localized by a retention mechanism that prevents their forward progress. Nevertheless, some early Golgi proteins acquire late Golgi modifications. Herein, we describe GPP130 (Golgi phosphoprotein of 130 kDa), a 130-kDa phosphorylated and glycosylated integral membrane protein localized to the cis/medial Golgi. GPP130 appears to be the human counterpart of rat Golgi integral membrane protein, cis (GIMPc), a previously identified early Golgi antigen that acquires late Golgi carbohydrate modifications. The sequence of cDNAs encoding GPP130 indicate that it is a type II membrane protein with a predicted molecular weight of 81,880 and an unusually acidic lumenal domain. On the basis of the alignment with several rod-shaped proteins and the presence of multiple predicted coiled-coil regions, GPP130 may form a flexible rod in the Golgi lumen. In contrast to the behavior of previously studied type II Golgi proteins, overexpression of GPP130 led to a pronounced accumulation in endocytotic vesicles, and endogenous GPP130 reversibly redistributed to endocytotic vesicles after chloroquine treatment. Thus, localization of GPP130 to the early Golgi involves steps that are saturable and sensitive to lumenal pH, and GPP130 contains targeting information that specifies its return to the Golgi after chloroquine washout. Given that GIMPc acquires late Golgi modifications in untreated cells, it seems likely that GPP130/GIMPc continuously cycles between the early Golgi and distal compartments and that an unidentified retrieval mechanism is important for its targeting.

GA2/GM2/GD2 synthase localizes to the trans-Golgi network of CHO-K1 cells

“…On the contrary, human galactosyltransferase is found located in the distal Golgi when overexpressed 2-4-fold in L cells, or in the medial-Golgi or the ER when overexpressed in COS-7 cells [37]. On the other hand, overexpression of GPP130, a cis\medial-Golgi protein, leads to its partial mislocalization to endocytotic vesicles [38]. In the case of GalNAc-T, in no clone have we observed localization of the enzyme to post-Golgi structures.…”

Identification of a gain-of-function mutation in a Golgi P-type ATPase that enhances Mn 2+ efflux and protects against toxicity

“…Cell culture, transfections, immunofluorescence microscopy, image analysis, and immunoblot analyses were done as described by us previously (15,19,20,40,41). Tertiary structure of SPCA1 was derived using the algorithms of the MODBASE server (http://modbase.compbio.ucsf.edu/modbase-cgi/index.…”

Proteomics of Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC) Membranes from Brefeldin A-treated HepG2 Cells Identifies ERGIC-32, a New Cycling Protein That Interacts with Human Erv46

“…Antibodies-Mouse monoclonal antibodies used were: G1/93 against ERGIC-53 (5), G1/221 against transferrin receptor (8), H4A3 against LAMP-1 (37) (Developmental Studies Hybridoma Bank, University of Iowa), G1/296 against CLIMP-63 (p63) (38), G1/133 against giantin (39), A1/118 against GPP130 (40), A1/182/5 against BAP31 (7), a mAb raised against a cytoplasmic peptide of the KDEL receptor (35), 12CA5 against the HA epitope, 9E10 against the Myc epitope. Rabbit polyclonal antibodies were used against the following proteins: Erv46 (41), TGN46 (42) (kind gift of S. Ponnambalam, University of Leeds), KDEL-R (43) (kind gift of H.-D. Söling, Max-Planck-Institut fü r Biophysikalische Chemie, Göttingen, Germany), Sec31p (44) (kind gift of F. Gorelick, Yale University), ␤-COP (kind gift of S. Pfeffer, Stanford University), and receptor-associated protein, RAP (Bu et al (71)).…”