Paul A. Gleeson

Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking.

The Menkes P‐type ATPase (MNK), encoded by the Menkes gene (MNK; ATP7A), is a transmembrane copper‐translocating pump which is defective in the human disorder of copper metabolism, Menkes disease. Recent evidence that the MNK P‐type ATPase has a role in copper efflux has come from studies using copper‐resistant variants of cultured Chinese hamster ovary (CHO) cells. These variants have MNK gene amplification and consequently overexpress MNK, the extents of which correlate with the degree of elevated copper efflux. Here, we report on the localization of MNK in these copper‐resistant CHO cells when cultured in different levels of copper. Immunofluorescence studies demonstrated that MNK is predominantly localized to the Golgi apparatus of cells in basal medium. In elevated copper conditions there was a rapid trafficking of MNK from the Golgi to the plasma membrane. This shift in steady‐state distribution of MNK was reversible and not dependent on new protein synthesis. In media containing basal copper, MNK accumulated in cytoplasmic vesicles after treatment of cells with a variety of agents that inhibit endosomal recycling. We suggest that MNK continuously recycles between the Golgi and the plasma membrane and elevated copper shifts the steady‐state distribution from the Golgi to the plasma membrane. These data reveal a novel system of regulated protein trafficking which ultimately leads to the efflux of an essential yet potentially toxic ligand, where the ligand itself appears directly and specifically to stimulate the trafficking of its own transporter.

Macropinocytosis: an endocytic pathway for internalising large gulps

Macropinocytosis is a regulated form of endocytosis that mediates the non‐selective uptake of solute molecules, nutrients and antigens. It is an actin‐dependent process initiated from surface membrane ruffles that give rise to large endocytic vacuoles called macropinosomes. Macropinocytosis is important in a range of physiological processes; it is highly active in macrophages and dendritic cells where it is a major pathway for the capture of antigens, it is relevant to cell migration and tumour metastasis and it represents a portal of cell entry exploited by a range of pathogens. The molecular basis for the formation and maturation of macropinosomes has only recently begun to be defined. Here, we review the general characteristics of macropinocytosis, describe some of the regulators of this pathway, which have been identified to date and highlight strategies to explore the relevance of this endocytosis pathway in vivo.

C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking

Intronic expansion of a hexanucleotide GGGGCC repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene is the major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. However, the cellular function of the C9ORF72 protein remains unknown. Here, we demonstrate that C9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictions that C9ORF72 bears Rab guanine exchange factor activity. Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin from the plasma membrane to Golgi apparatus, internalization of TrkB receptor and altered the ratio of autophagosome marker light chain 3 (LC3) II:LC3I, indicating that C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence that C9ORF72 regulates autophagy. Investigation of proteins interacting with C9ORF72 using mass spectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking.

Organ‐specific Autoimmunity Induced by Lymphopenia

In 1973 two groups reported the induction of organ-specific autoimmune diseases by T cell deprivation. Penhale and co-workers (Penhale et al. 1973) found that autoimmune thyroiditis could be induced in 5-week-old Wistar rats by thymeclomy and repeated low-dose irradiation. On the other hand, Nishizuka and colleagues (Nishizuka et al. 1973) made the fascinating observation that autoimmune thyroiditis spontaneously developed in mice following thymectomy alone, so long as the thymectomy was carried out in the neonatal period. Subsequently, neonatal thymectomy was shown to induce a wide variety of organ-specific autoimmune diseases in mice, with different strains showing differences in disease susceptibility. The overlap in autoimmune diseases in this mouse model is reminiscent of the overlap observed in the endocrine organ/stomach cluster of organ-specific autoimmune diseases of humans. From 1975 onwards a number of other manipulations of the immune system have been shown to induce organ-specific autoimmune diseases in rodents. A common characteristic of these manipulations is either a deficit of T cells or reduction of the TCR repertoire, in other words functional T cell lymphopenia. The range of autoimmune diseases induced by these manipulations is most impressive and includes diabetes, thyroditis, gastritis, orchitis, oophoritis, prostatitis, and coagulating adenitis. Many of these diseases are rare in laboratory strains of rats and mice. Evidence that the autoimmune diseases are due to specific immune responses is supported by the presence of infiltrating lymphocytes in the target organ, circulat-

The Phox Homology (PX) Domain-dependent, 3-Phosphoinositide-mediated Association of Sorting Nexin-1 with an Early Sorting Endosomal Compartment Is Required for Its Ability to Regulate Epidermal Growth Factor Receptor Degradation

Recent studies have shown that phox homology (PX) domains act as phosphoinositide-binding motifs. The majority of PX domains studied show binding to phosphatidylinositol 3-monophosphate (PtdIns(3)P), an association that allows the host protein to localize to membranes of the endocytic pathway. One issue, however, is whether PX domains may have alternative phosphoinositide binding specificities that could target their host protein to distinct subcellular compartments or allow their allosteric regulation by phosphoinositides other than PtdIns(3)P. It has been reported that the PX domain of sorting nexin 1 (SNX1) specifically binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) (Zhong, Q., Lazar, C. S., Tronchere, H., Sato, T., Meerloo, T., Yeo, M., Songyang, Z., Emr, S. D., and Gill, G. N. (2002)Proc. Natl. Acad. Sci. U. S. A. 99, 6767–6772). In the present study, we have shown that whereas SNX1 binds PtdIns(3,4,5)P3 in protein:lipid overlay assays, in liposomes-based assays, binding is observed to PtdIns(3)P and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) but not to PtdIns(3,4,5)P3. To address the significance of PtdIns(3,4,5)P3 binding, we examined the subcellular localization of SNX1 under conditions in which plasma membrane PtdIns(3,4,5)P3 levels were significantly elevated. Under these conditions, we failed to observe association of SNX1 with this membrane. However, consistent with the binding to PtdIns(3)P and PtdIns(3,5)P2 being of more physiological significance was the observation that the association of SNX1 with an early endosomal compartment was dependent on a 3-phosphoinositide-binding PX domain and the presence of PtdIns(3)P on this compartment. Finally, we have shown that the PX domain-dependent/early endosomal association of SNX1 is important for its ability to regulate the targeting of internalized epidermal growth factor receptor for lysosomal degradation.

A large family of endosome-localized proteins related to sorting nexin 1

Sorting nexin 1 (SNX1), a peripheral membrane protein, has previously been shown to regulate the cell-surface expression of the human epidermal growth factor receptor [Kurten, Cadena and Gill (1996) Science 272, 1008-1010]. Searches of human expressed sequence tag databases with SNX1 revealed eleven related human cDNA sequences, termed SNX2 to SNX12, eight of them novel. Analysis of SNX1-related sequences in the Saccharomyces cerevisiae genome clearly shows a greatly expanded SNX family in humans in comparison with yeast. On the basis of the predicted protein sequences, all members of this family of hydrophilic molecules contain a conserved 70-110-residue Phox homology (PX) domain, referred to as the SNX-PX domain. Within the SNX family, subgroups were identified on the basis of the sequence similarities of the SNX-PX domain and the overall domain structure of each protein. The members of one subgroup, which includes human SNX1, SNX2, SNX4, SNX5 and SNX6 and the yeast Vps5p and YJL036W, all contain coiled-coil regions within their large C-terminal domains and are found distributed in both membrane and cytosolic fractions, typical of hydrophilic peripheral membrane proteins. Localization of the human SNX1 subgroup members in HeLa cells transfected with the full-length cDNA species revealed a similar intracellular distribution that in all cases overlapped substantially with the early endosome marker, early endosome autoantigen 1. The intracellular localization of deletion mutants and fusions with green fluorescent protein showed that the C-terminal regions of SNX1 and SNX5 are responsible for their endosomal localization. On the basis of these results, the functions of these SNX molecules are likely to be unique to endosomes, mediated in part by interactions with SNX-specific C-terminal sequences and membrane-associated determinants.

Gastric H+, K+-adenosine triphosphatase β subunit is required for normal function, development, and membrane structure of mouse parietal cells

BACKGROUND & AIMS Parietal cells of the gastric mucosa contain a complex and extensive secretory membrane system that harbors gastric H(+),K(+)-adenosine triphosphatase (ATPase), the enzyme primarily responsible for acidification of the gastric lumen. We have produced mice deficient in the H(+),K(+)-ATPase beta subunit to determine the role of the protein in the biosynthesis of this membrane system and the biology of gastric mucosa. METHODS Mice deficient in the H(+), K(+)-ATPase beta subunit were produced by gene targeting. RESULTS The stomachs of H(+),K(+)-ATPase beta subunit-deficient mice were achlorhydric. Histological and immunocytochemical analyses with antibodies to the H(+),K(+)-ATPase alpha subunit revealed that parietal cell development during ontogeny was retarded in H(+), K(+)-ATPase beta subunit-deficient mice. In 15-day-old mice, cells with secretory canaliculi were observed in wild-type but not in H(+), K(+)-ATPase beta subunit-deficient mice. Parietal cells of H(+), K(+)-ATPase beta subunit-deficient mice 17 days and older contained an abnormal canaliculus that was dilated and contained fewer and shorter microvilli than normal. In older parietal cells, the abnormal canaliculus was massive (25 micrometer in diameter) and contained few microvilli. We did not observe typical tubulovesicular membranes in any parietal cell from H(+),K(+)-ATPase beta subunit-deficient mice. Histopathologic alterations were only observed in the stomach. CONCLUSIONS The H(+),K(+)-ATPase beta subunit is required for acid-secretory activity of parietal cells in vivo, normal development and cellular homeostasis of the gastric mucosa, and attainment of the normal structure of the secretory membranes.

Intracellular sorting and transport of proteins

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, integrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations.

The trans‐Golgi Network Golgin, GCC185, is Required for Endosome‐to‐Golgi Transport and Maintenance of Golgi Structure

Four mammalian golgins are specifically targeted to the trans‐Golgi network (TGN) membranes via their C‐terminal GRIP domains. The TGN golgins, p230/golgin‐245 and golgin‐97, are recruited via the GTPase Arl1, whereas the TGN golgin GCC185 is recruited independently of Arl1. Here we show that GCC185 is localized to a region of the TGN distinct from Arl1 and plays an essential role in maintaining the organization of the Golgi apparatus. Using both small interfering RNA (siRNA) and microRNA (miRNA), we show that depletion of GCC185 in HeLa cells frequently resulted in fragmentation of the Golgi apparatus. Golgi apparatus fragments were dispersed throughout the cytoplasm and contained both cis and trans markers. Trafficking of anterograde and retrograde cargo was analysed over an extended period following GCC185 depletion. Early effects of GCC185 depletion included a perturbation in the distribution of the mannose‐6‐phosphate receptor and a block in shiga toxin trafficking to the Golgi apparatus, which occurred in parallel with the fragmentation of the Golgi ribbon. Internalized shiga toxin accumulated in Rab11‐positive endosomes, indicating GCC185 is essential for transport between the recycling endosome and the TGN. In contrast, the plasma membrane–TGN recycling protein TGN38 was efficiently transported into GCC185‐depleted Golgi apparatus fragments throughout a 96‐h period, and anterograde transport of E‐cadherin was functional until a late stage of GCC185 depletion. This study demonstrated (i) a more effective long‐term depletion of GCC185 using miRNA than siRNA and (ii) a dual role for the GCC185 golgin in the regulation of endosome‐to‐TGN membrane transport and in the organization of the Golgi apparatus.

E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by Golgin-97

E‐cadherin is a cell–cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane‐bound carriers for the post‐Golgi exocytosis of E‐cadherin have not been characterized. Green fluorescent protein (GFP)‐tagged E‐cadherin (Ecad‐GFP) is transported from the trans‐Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. Here, we examine the association of golgins with tubular carriers containing E‐cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full‐length proteins and were coexpressed with Ecad‐GFP. The GRIP domains of p230/golgin‐245 and golgin‐97 had overlapping but nonidentical distributions on the TGN; both domains were on TGN‐derived tubules but only the golgin‐97 GRIP domain coincided with Ecad‐GFP tubules in live cells. When the Arl1‐binding endogenous golgins, p230/golgin‐245 and golgin‐97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad‐GFP was inhibited. siRNA knockdown of golgin‐97 also inhibited trafficking of Ecad‐GFP. Thus, the GRIP domains of p230/golgin‐245 and golgin‐97 bind discriminately to distinct membrane subdomains of the TGN. Golgin‐97 is identified as a selective and essential component of the tubulovesicular carriers transporting E‐cadherin out of the TGN.