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.

Golgi inheritance during cell division involves Golgi disassembly but it remains unclear whether the breakdown product is dispersed vesicles, clusters of vesicles or a fused ER/Golgi network. Evidence against the fused ER/Golgi hypothesis was previously obtained from subcellular fractionation studies, but left concerns about the means used to obtain and disrupt mitotic cells. Here, we performed velocity gradient analysis on otherwise untreated cells shaken from plates 9 h after release from an S-phase block. In addition, we used digitonin and freeze/thaw permeabilization as alternatives to mechanical homogenization. Under each of these conditions, approximately 75% of the Golgi was recovered in a population of small vesicles that lacked detectable ER. We also used multilabel fluorescent microscopy with optical sectioning by deconvolution to compare the 3D metaphase staining pattern of endogenous Golgi and ER markers. Although both ER and Golgi staining were primarily diffuse, only the ER was excluded from the mitotic spindle region. Surprisingly, only 2% of the Golgi fluorescence was present as resolvable structures previously characterized as vesicle clusters. These were not present in the ER pattern. Significantly, a portion of the diffuse Golgi fluorescence, presumably representing dispersed 60-nm vesicles, underwent an apparent rapid aggregation with the larger Golgi structures upon treatments that impaired microtubule integrity. Therefore, mitotic Golgi appears to be in a dynamic equilibrium between clustered and free vesicles, and accurate partitioning may be facilitated by microtubule-based motors acting on the clusters to insure random and uniform distribution of the vesicles.

By forming a molecular tether between two membranes, p115, giantin, and GM130 may mediate multiple Golgi-related processes including vesicle transport, cisternae formation, and cisternal stacking. The tether is proposed to involve the simultaneous binding of p115 to giantin on one membrane and to GM130 on another membrane. To explore this model, we tested for the presence of the putative giantin-p115-GM130 ternary complex. We first mapped p115-binding site in giantin to a 70-amino acid coiled-coil domain at the extreme N terminus, a position that may exist up to 400 nm away from the Golgi membrane. We then generated glutathione Stransferase (GST) fusion proteins containing either giantin's or GM130's p115 binding site and tested whether such proteins could bind p115 and GM130 or bind p115 and giantin, respectively. Unexpectedly, GST fusions containing either the giantin or the GM130 p115 binding site efficiently bound p115, but the p115 bound to GSTgiantin did not bind GM130, and the p115 bound to GST-GM130 did not bind giantin. To explain this result, we mapped the giantin binding site in p115 and found that it is located at the C-terminal acidic domain, the same domain involved in binding GM130. The presence of a single binding site in p115 for giantin and GM130 was confirmed by demonstration that giantin and GM130 compete for binding to p115. These results question a simple tethering model involving a ternary giantinp115-GM130 complex and suggest that p115-giantin and p115-GM130 interactions might mediate independent membrane tethering events.