The Function of the Intermediate Compartment in Pre-Golgi Trafficking Involves its Stable Connection with the Centrosome

Abstract

Because the functional borders of the intermediate compartment (IC) are not well defined, the spatial map of the transport machineries operating between the endoplasmic reticulum (ER) and the Golgi apparatus remains incomplete. Our previous studies showed that the IC consists of interconnected vacuolar and tubular parts with specific roles in pre-Golgi trafficking. Here, using live cell imaging, we demonstrate that the tubules containing the GTPase Rab1A create a long-lived membrane compartment around the centrosome. Separation of this pericentrosomal domain of the IC from the Golgi ribbon, due to centrosome motility, revealed that it contains a distinct pool of COPI coats and acts as a temperaturesensitive way station in post-ER trafficking. However, unlike the Golgi, the pericentrosomal IC resists the disassembly of COPI coats by brefeldin A, maintaining its juxtaposition with the endocytic recycling compartment, and operation as the focal point of a dynamic tubular network that extends to the cell periphery. These results provide novel insight into the compartmental organization of the secretory pathway and Golgi biogenesis. Moreover, they reveal a direct functional connection between the IC and the endosomal system, which evidently contributes to unconventional transport of the cystic fibrosis transmembrane conductance regulator to the cell surface. INTRODUCTIONThe biosynthetic-secretory pathway sorts and delivers newly synthesized proteins and lipids, many with attached glycans, to the various endomembrane compartments of the cell and to its exterior (Palade, 1982). The dynamics and spatial organization of the secretory compartments-endoplasmic reticulum (ER), intermediate compartment (IC), and Golgi apparatus-depend on the microtubule (MT) and actin cytoskeleton and associated motor proteins (Thyberg and Moskalewski, 1999;Lippincott-Schwartz et al., 2000;Allan et al., 2002;Egea et al., 2006). The prevailing paradigm of the secretory pathway in mammalian cells states that the Golgi apparatus, positioned around the MT-organizing center (MTOC)/centrosome (Rios and Bornens, 2003), receives nascent proteins from widely distributed ER exit sites (ERES) due to MT-dependent centralization of IC elements (Saraste and Svensson, 1991;Bannykh et al., 1996;Presley et al., 1997;Scales et al., 1997;Hammond and Glick, 2000). After their post-translational modification and sorting in the Golgi, the proteins are distributed to the endolysosomal system, secretory granules, and the plasma membrane (PM;De Matteis and Luini, 2008). This spatial arrangement of the secretory process, consisting of centripetal and centrifugal transfer of biosynthetic products, is not essential for transport as such (Thyberg and Moskalewski, 1999;Hawes et al., 2008). However, it can promote the coalignment of the secretory and endocytic routes, allowing their communication at multiple sites (Saraste and Kuismanen, 1992;Bonifacino and Rojas, 2006) and facilitate the processing and sorting of cargo along these pathways via the establishment of a lume...

Division of the intermediate compartment at the onset of mitosis provides a mechanism for Golgi inheritance

“…In addition, they undergo saltatory movements to the spindle poles ( Fig. 4C,D; supplementary material Movies 5, 6) at speeds (,1 mm/sec) similar to those recorded during interphase Sannerud et al, 2006;Marie et al, 2009), suggesting that they occur in a dynein-dependent fashion (Waterman-Storer et al, 1993;Brownhill et al, 2009). Furthermore, the pcIC elements show motility as they position at a fixed distance from the spindle poles (supplementary material Movie 7).…”

“…Notably, the observed partial overlap of b-COP and Rab1A (Fig. 7A) indicates that the division of the IC into vacuolar and tubular subdomains, defined by these components (Horstmann et al, 2002;Sannerud et al, 2006;Marie et al, 2009), persists in metaphasic cells. Removal of the coats by BFA did not abolish the IC elements, showing that they maintain their drug-resistant character ) during mitosis (Fig.…”

“…The movements of Rab1A-positive tubules between more stationary peripheral IC clusters and the pcIC, that take place along MT tracks, are the hallmarks of IC dynamics in interphasic NRK cells (Sannerud et al, 2006;Marie et al, 2009). Since the mitotic IC maintains its connection to the spindle apparatus, it was of interest to employ spinning disk CM to study its dynamic properties.…”

“…However, the pleiomorphic vacuolar domains of the IC that bud COPI vesicles (Martínez-Menárguez et al, 1999;Horstmann et al, 2002;Sannerud et al, 2006) also extend tubules enriched in the GTPase Rab1A (Palokangas et al, 1998;Sannerud et al, 2006). Moreover, in vivo imaging of Rab1A revealed that the tubules form an extensive, interconnected network, which is stably anchored at the cell centre via its pericentrosomal domain (pcIC; Marie et al, 2009;Saraste et al, 2009), encouraging the reinvestigation of the mitotic fate of the IC. Here we show that the pcIC expands and divides at the onset of mitosis in a process that is intimately linked to centrosome motility and separation, as well as the fragmentation of the Golgi ribbon.…”

The Role of Glycans in Apical Sorting of Proteins in Polarized Epithelial Cells

“…In mammalian cells, the intermediate compartment (IC), consisting of vacuolar and tubular membrane domains and functionally positioned between the ER and the Golgi apparatus, is also stably localized in a perinuclear position at the cell center, close to the Golgi cisternae. Although the trans region of the Golgi apparatus has been regarded as the primary sorting area for proteins and lipids in the secretory pathway, the IC has been suggested as a protein and lipid sorting station operating prior to entry into the Golgi apparatus (47)(48)(49). In fact, lateral segregation of proteins in the secretory pathway can occur already in the ER (50-53, 3, 18).…”