George Banting

BST-2/HM1.24 is a raft-associated apical membrane protein with an unusual topology

An expression screen of a rat cDNA library for sequences encoding Golgi‐localized integral membrane proteins identified a protein with an apparent novel topology, i.e. with both an N‐terminal transmembrane domain and a C‐terminal glycosyl‐phosphatidylinositol (GPI) anchor. Our data are consistent with this. Thus, the protein would have a topology that, in mammalian cells, is shared only by a minor, but pathologically important, topological isoform of the prion protein (PrP). The human orthologue of this protein has been described previously (BST‐2 or HM1.24 antigen) as a cell surface molecule that appears to be involved in early pre‐B‐cell development and which is present at elevated levels at the surface of myeloma cells. We show that rat BST‐2/HM1.24 has both a cell surface and an intracellular (juxtanuclear) location and is efficiently internalized from the cell surface. We also show that the cell surface pool of BST‐2/HM1.24 is predominantly present in the apical plasma membrane of polarized cells. The fact that rat BST‐2/HM1.24 apparently possesses a GPI anchor led us to speculate that it might exist in cholesterol‐rich lipid microdomains (lipid rafts) at the plasma membrane. Data from several experiments are consistent with this localization. We present a model in which BST‐2/HM1.24 serves to link adjacent lipid rafts within the plasma membrane.

HIV-1 Antagonism of CD317 Is Species Specific and Involves Vpu-Mediated Proteasomal Degradation of the Restriction Factor

Mammals encode proteins that inhibit viral replication at the cellular level. In turn, certain viruses have evolved genes that can functionally counteract these intrinsic restrictions. Human CD317 (BST-2/HM1.24/tetherin) is a restriction factor that blocks release of human immunodeficiency virus type 1 (HIV-1) from the cell surface and can be overcome by HIV-1 Vpu. Here, we show that mouse and rat CD317 potently inhibit HIV-1 release but are resistant to Vpu. Interspecies chimeras reveal that the rodent-specific resistance and human-specific sensitivity to Vpu antagonism involve all three major structural domains of CD317. To promote virus release, Vpu depletes cellular pools of human CD317, but not of the rodent orthologs, by accelerating its degradation via the 20S proteasome. Thus, HIV-1 Vpu suppresses the expression of the CD317 antiviral factor in human cells, and the species-specific resistance to this suppression may guide the development of small animal models of HIV infection.

A CD317/tetherin–RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

CD317/tetherin is a lipid raft–associated integral membrane protein with a novel topology. It has a short N-terminal cytosolic domain, a conventional transmembrane domain, and a C-terminal glycosyl-phosphatidylinositol anchor. We now show that CD317 is expressed at the apical surface of polarized epithelial cells, where it interacts indirectly with the underlying actin cytoskeleton. CD317 is linked to the apical actin network via the proteins RICH2, EBP50, and ezrin. Knocking down expression of either CD317 or RICH2 gives rise to the same phenotype: a loss of the apical actin network with concomitant loss of apical microvilli, an increase in actin bundles at the basal surface, and a reduction in cell height without any loss of tight junctions, transepithelial resistance, or the polarized targeting of apical and basolateral membrane proteins. Thus, CD317 provides a physical link between lipid rafts and the apical actin network in polarized epithelial cells and is crucial for the maintenance of microvilli in such cells.

Regions of human kidney anion exchanger 1 (kAE1) required for basolateral targeting of kAE1 in polarised kidney cells: mis-targeting explains dominant renal tubular acidosis (dRTA)

Distal renal tubular acidosis (dRTA) is characterised by defective acid secretion by kidney α-intercalated cells. Some dominantly inherited forms of dRTA result from anion exchanger 1 (AE1) mutations. We have developed a stably transfected cell model for the expression of human kidney AE1 (kAE1) and mutant kAE1 proteins in MDCKI cells. Normal kAE1 was delivered to the plasma membrane of non-polarised cells and to the basolateral membrane of polarised cells. The AE1 N-glycan was processed to a complex form. Surprisingly, expression of kAE1 increased the permeability of the paracellular barrier of polarised MDCKI monolayers. All dominant dRTA mutations examined altered the targeting of kAE1 in MDCKI cells. The mutant proteins kAE1(R589H), kAE1(S613F) and kAE1(R901Stop) were retained in the ER in non-polarised cells, but the kAE1(R901Stop) protein was also present in late endosomes/lysosomes. The complex N-glycan of kAE1(R901Stop) was larger than that of normal kAE1. In polarised cells, the mutant kAE1(R901Stop) was mis-targeted to the apical membrane, while the kAE1(R589H) and kAE1(S613F) mutants did not reach the cell surface. These results demonstrate that dominant dRTA mutations cause aberrant targeting of kAE1 in polarised kidney cells and provide an explanation for the origin of dominant dRTA. Our data also demonstrate that the 11 C-terminal residues of kAE1 contain a tyrosine-dependent basolateral targeting signal that is not recognised by μ1B-containing AP-1 adaptor complexes. In the absence of the N-terminus of kAE1, the C-terminus was not sufficient to localise kAE1 to the basolateral membrane. These results suggest that a determinant within the kAE1 N-terminus co-operates with the C-terminus for kAE1 basolateral localisation.

Tyrphostin A23 inhibits internalization of the transferrin receptor by perturbing the interaction between tyrosine motifs and the medium chain subunit of the AP-2 adaptor complex

Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs within the cytosolic domains of integral membrane proteins. Many such motifs conform to the consensus YXXΦ, where Φ represents a bulky hydrophobic residue. This motif interacts with the medium chain (μ) subunits of adaptor complexes that link the cytosolic domains of integral membrane proteins to the clathrin coat involved in vesicle formation. The YXXΦ motif is similar to motifs in which the tyrosine residue is phosphorylated by tyrosine kinases. Tyrphostins (structural analogs of tyrosine) are inhibitors of tyrosine kinases and function by binding to the active sites of the enzymes. We previously showed that, in vitro and in yeast two-hybrid interaction assays, some tyrphostins can inhibit the interaction between YXXΦ motifs and the μ2 subunit of the AP-2 adaptor complex (Crump, C., Williams, J. L., Stephens, D. J., and Banting, G. (1998) J. Biol. Chem. 273, 28073–28077). A23 is such a tyrphostin. We now show that molecular modeling of tyrphostin A23 into the tyrosine-binding pocket in μ2 provides a structural explanation for A23 being able to inhibit the interaction between YXXΦ motifs and μ2. Furthermore, we show that A23 inhibited the internalization of125I-transferrin in Heb7a cells without having any discernible effect on the morphology of compartments of the endocytic pathway. Control tyrphostins, active as inhibitors of tyrosine kinase activity, but incapable of inhibiting the YXXΦ motif/μ2 interaction, did not inhibit endocytosis. These data are consistent with A23 inhibition of the YXXΦ motif/μ2 interaction in intact cells and with the possibility that different tyrphostins may be used to inhibit specific membrane trafficking events in eukaryotic cells.

Hippocalcin Functions as a Calcium Sensor in Hippocampal LTD

It is not fully understood how NMDAR-dependent LTD causes Ca(2+)-dependent endocytosis of AMPARs. Here we show that the neuronal Ca(2+) sensor hippocalcin binds the beta2-adaptin subunit of the AP2 adaptor complex and that along with GluR2 these coimmunoprecipitate in a Ca(2+)-sensitive manner. Infusion of a truncated mutant of hippocalcin (HIP(2-72)) that lacks the Ca(2+) binding domains prevents synaptically evoked LTD but has no effect on LTP. These data indicate that the AP2-hippocalcin complex acts as a Ca(2+) sensor that couples NMDAR-dependent activation to regulated endocytosis of AMPARs during LTD.

CK2 and GAK/auxilin2 Are Major Protein Kinases in Clathrin-Coated Vesicles

Several peripheral membrane proteins associated with clathrin‐coated vesicles (CCVs) are reversibly phosphorylated, but it is not clear precisely which protein kinases are involved. In order to address this question directly, we have isolated highly purified CCVs from porcine brain. The peripheral membrane proteins have been removed and assayed for kinase activity using the CCV peripheral membrane proteins as substrate. The major kinase activity identified has a molecular mass of 40 kDa, is inhibited by known specific inhibitors of the protein kinase CK2 and is recognised by an antibody specific to CK2. We show that CK2 is responsible for the phosphorylation of the majority of CCV‐associated proteins that are subject to phosphorylation. Intriguingly, CK2 is inactive when associated with CCVs but becomes active once the clathrin coat has been removed. The medium subunit of the AP2 adaptor complex (μ2) is not a substrate for CK2, but is phosphorylated by a second kinase that we show to be cyclin G‐associated kinase (GAK/auxilin2). Unlike the situation for the CK2 substrates, μ2 is a substrate for GAK/auxilin2, both in intact CCVs and in solution. In addition, we show that the ‘stripped’ CCV membranes that remain once the peripheral membrane proteins have been removed from CCVs inhibit CK2 but not GAK/auxilin2 activity.

Intracellular targetting signals of polymeric immunoglobulin receptors are highly conserved between species

A rat liver cDNA library, constructed in the plasmid expression vector pUEX, was immunoscreened using a rabbit polyclonal antiserum raised against rat liver Golgi membrane proteins. A sub‐set of isolated clones were shown to encode the rat polymeric immunoglobulin receptor (pIgR). DNA sequence analysis of these clones provided the complete coding sequence of rat pIgR. Subsequent alignment of rat, rabbit and human predicted amino acid sequences demonstrated that the greatest degree of homology between the three pIgRs lies in their cytoplasmic tails; a region previously shown to be important for correct targetting and trancytosis of rabbit pIgR [(1984) Nature 308, 37–43].

Vacuolar ATPase inactivation blocks recycling to the trans-Golgi network from the plasma membrane

TGN38/41 is an integral membrane protein which recycles between the trans‐Golgi network (TGN) and the cell surface but is predominantly located in the TGN of rat (NRK) cells at steady state. As part of our studies on the mechanism and route of recycling between the TGN and the cell surface we have used chloroquine or Bafilomycin A1 to modulate the lumenal pH of endocytic organelles. The data we present demonstrate that inactivation of the proton pump which maintains the acidic environment within the lumen of endocytic organelles leads to an accumulation of TGN38/41 in early endosomes. These data confirm the observation that TGN38/41 recycles between the plasma membrane and the TGN and identifies a specific block in that recycling pathway.

A PtdIns(3)P-specific probe cycles on and off host cell membranes during Salmonella invasion of mammalian cells

Salmonella invade nonphagocytic cells by eliciting their own internalization; upon contact with the host cell, the bacteria induce membrane ruffles highly localized to the point of contact between the invading bacterium and the host cell. The bacterium is then internalized into an unusual cytosolic organelle, the Salmonella-containing vacuole (SCV). Early endosomal markers (including EEA1) have recently been shown to be associated with the SCV shortly after invasion. EEA1, a protein involved in early endosome fusion, is recruited to early endosomal membranes in part by the interaction between its FYVE finger and phosphatidylinositol 3-phosphate [PtdIns(3)P], a characteristic lipid of early endosomes. This suggests a possible role for PtdIns(3)P during Salmonella infection. To investigate this, we generated a highly specific probe for PtdIns(3)P that was used to follow invasion of Salmonella in nonphagocytic cells. Here, we show that PtdIns(3)P is present on the membranes of SCVs shortly after invasion and also that it is present on the membrane ruffles produced immediately prior to invasion. We also show that this specific probe cycles on and off the membranes of nascent SCVs even when PtdIns 3-kinase activity is inhibited, demonstrating that invading Salmonella influence the composition of the membranes that envelop them during invasion.