Overexpression of a novel sorting nexin, SNX15, affects endosome morphology and protein trafficking

Sorting nexin (SNX) 15 is a novel member of the SNX family of proteins. Although the functions of most SNXs have not yet been determined, several family members (e.g., SNX1, SNX2, SNX3, and SNX8) are orthologs of yeast proteins involved in protein trafficking. Overexpression of myc-tagged SNX15 in COS-7 cells altered the morphology of several endosomal compartments. In transient transfection experiments, myc-SNX15 was first seen in small punctate spots and small ring structures. Later, myc-SNX15 was found in larger rings. Finally, myc-SNX15 was observed in large, amorphous membrane-limited structures. These structures contained proteins from lysosomes, late endosomes, early endosomes, and the trans-Golgi network. However, the morphology of the endoplasmic reticulum and Golgi was not affected by overexpression of myc-SNX15. In myc-SNX15-overexpressing cells, the endocytosis of transferrin was severely inhibited and endocytosis of tac-trans-Golgi network (TGN) 38 and tac-furin was slowed. In addition, the recycling of internalized tac-TGN38 and tac-furin was also inhibited. Both the morphological and biochemical data indicate that SNX15 plays a crucial role in trafficking through the endocytic pathway. This is the first demonstration that a mammalian SNX protein is involved in protein trafficking.     

Annexin VI stimulates endocytosis and is involved in the trafficking of low density lipoprotein to the prelysosomal compartment

Annexins are calcium-binding proteins with a wide distribution in most polarized and nonpolarized cells that participate in a variety of membrane-membrane interactions. At the cell surface, annexin VI is thought to remodel the spectrin cytoskeleton to facilitate budding of coated pits. However, annexin VI is also found in late endocytic compartments in a number of cell types, indicating an additional important role at later stages of the endocytic pathway. Therefore overexpression of annexin VI in Chinese hamster ovary cells was used to investigate its possible role in endocytosis and intracellular trafficking of low density lipoprotein (LDL) and transferrin. While overexpression of annexin VI alone did not alter endocytosis and degradation of LDL, coexpression of annexin VI and LDL receptor resulted in an increase in LDL uptake with a concomitant increase of its degradation. Whereas annexin VI showed a wide intracellular distribution in resting Chinese hamster ovary cells, it was mainly found in the endocytic compartment and remained associated with LDL-containing vesicles even at later stages of the endocytic pathway. Thus, data presented in this study suggest that after stimulating endocytosis at the cell surface, annexin VI remains bound to endocytic vesicles to regulate entry of ligands into the prelysosomal compartment.     

Hrs and hbp: possible regulators of endocytosis and exocytosis

The molecular mechanisms of endocytosis and exocytosis are not yet fully understood. Hrs and Hbp, two tightly associated proteins in eukaryotic cells, have been implicated in these cellular processes. Hrs is homologous to Vps27p, an endosomal protein required for vacuolar and endocytic trafficking in yeast. Hrs is localized to early endosomes and is required for the normal morphology of early endosomes in mammalian cells. Hrs also associates with proteins implicated in endocytosis and exocytosis such as SNAP-25 and Eps15. Hrs treatment inhibits neurotransmitter release in permeabilized neuronal cells and its overexpression inhibits internalization of transferrin. Overexpression of dominant-negative Hbp mutants inhibits ligand-induced downregulation of growth factor/receptor complexes and immunoglobulin E receptor-triggered degranulation of secretory granules in mast cells. These observations suggest an important role for the Hrs/Hbp protein complex in vesicular trafficking during endocytosis and exocytosis.     

GLUT4 and Transferrin Receptor Are Differentially Sorted Along the Endocytic Pathway in CHO Cells

The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750–12754.). In this study, we demonstrate that at 37°C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37°C, cell surface–labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15°C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37°C after accumulating labeled GLUT4 at 15°C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15°C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37°C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15° and 37°C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.     

Calmodulin modulates hepatic membrane polarity by protein kinase C-sensitive steps in the basolateral endocytic pathway

Membrane polarity is maintained by a complex intermingling of various trafficking pathways, including basolateral and apical endocytosis. The present work was undertaken to better define the role of basolateral endocytic transport in apical membrane homeostasis. When polarized HepG2 hepatoma cells were incubated with calmodulin antagonists, the cells lost their polarity, as reflected by an inhibition of lipid transport of a fluorescent sphingomyelin to the apical membrane and an impediment of its recycling to the basolateral membrane. Instead, an accumulation of the lipid in dilated early endosomal compartments was observed, presumably due to a frustration of vesiculation. Interestingly, lipid transport to the apical pole, lipid recycling to the basolateral membrane and cell polarity were reestablished, while dilated compartments disappeared, when the cells were simultaneously treated with specific inhibitors of protein kinase C (PKC). Consistently, following activation of PKC, extensive dilation/vacuolation of early sorting endosomes was observed, very similar as seen upon treatment with calmodulin antagonists. Thus, the results indicate that membrane trafficking at early steps of the basolateral endocytic pathway in HepG2 cells is regulated by an intricate interplay between calmodulin and PKC. This interference, although not affecting endocytosis as such, compromises cell polarity by impeding membrane trafficking from early endosomes to the apical membrane.     

A chloroquine-resistant Swiss 3T3 cell line with a defect in late endocytic acidification

To investigate the role of acidification in cell proliferation, several cell lines resistant to chloroquine were isolated with the expectation that some would express altered endocytic acidification. The preliminary characterization of one of these lines, CHL60-64, is described. In contrast to endocytic mutants described previously, the initial phase of endocytic acidification, as measured by transferrin acidification, is normal in this cell line. However, a difference in subsequent endocytic acidification was observed in CHL60-64. In the parental cells, internalized dextran was fully acidified to approximately pH 5.5 within 1 h. In CHL60-64, the pH in the endocytic compartment was only 6.1 after 1 h and remained as high as 5.8 for at least 4 h. After an 8-h incubation, the pH decreased to 5.5, indicating that the second phase of acidification is only slowed in CHL60-64, and not blocked. Consistent with this retarded acidification, ATP-dependent acidification in vitro (as measured by acridine orange accumulation) was reduced in both the lysosomal fraction and the endosomal fraction isolated from CHL60-64. A decrease in the in vivo rate of acridine orange accumulation after perturbation with amine was also observed. In addition to amine resistance and defective acidification, CHL60-64 was found to be resistant to vacuolation in the presence of chloroquine and ammonium chloride, and was resistant to ouabain. Further studies on this new class of endocytosis mutant, in combination with existing mutants, should help to clarify the mechanisms responsible for the regulation of endocytic acidification.     

Regulation of transferrin-induced endocytosis by wild-type and C282Y-mutant HFE in transfected HeLa cells

The hereditaryhemochromatosis protein HFE is known to complex with the transferrinreceptor; however, its function regarding endocytosis of transferrin isunclear. We performed patch-clamp capacitance measurements intransfected HeLa cells carrying wild-type or C282Y-mutant HFE cDNAunder the control of a tetracycline-sensitive promoter. Whole cellexperiments in cells with suppressed expression of wild-type HFErevealed a decrease in membrane capacitance, reflecting predominance ofendocytosis in the presence of transferrin. Cells overexpressingC282Y-mutant HFE displayed less intense capacitance decreases, whereasno significant decrease was observed in cells overexpressing wild-typeHFE. The formation of single endocytic vesicles in cells withsuppressed expression of wild-type HFE was greatly increased in thepresence of transferrin as revealed by cell-attached recordings.According to their calculated diameters, many of these vesiclescorresponded to clathrin-coated vesicles. These results suggest thatwild-type HFE negatively modulates the endocytic uptake of transferrin.This inhibitory effect is attenuated in cells expressing C282Y-mutantHFE. Time-resolved measurements of cell membrane capacitance provide apowerful tool to study transferrin-induced endocytosis in single cells.

     

Initial entry of IRAP into the insulin-responsive storage compartment occurs prior to basal or insulin-stimulated plasma membrane recycling

To examine the acquisition of insulin sensitivity after the initial biosynthesis of the insulin-responsive aminopeptidase (IRAP), 3T3-L1 adipocytes were transfected with an enhanced green fluorescent protein-IRAP (EGFP-IRAP) fusion protein. In the absence of insulin, IRAP was rapidly localized (1-3 h) to secretory membranes and retained in these intracellular membrane compartments with little accumulation at the plasma membrane. However, insulin was unable to induce translocation to the plasma membrane until 6-9 h after biosynthesis. This was in marked contrast to another type II membrane protein (syntaxin 3) that rapidly defaulted to the plasma membrane 3 h after expression. In parallel with the time-dependent acquisition of insulin responsiveness, the newly synthesized IRAP protein converted from a brefeldin A-sensitive to a brefeldin A-insensitive state. The initial trafficking of IRAP to the insulin-responsive compartment was independent of plasma membrane endocytosis, as expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis but had no effect on the insulin-stimulated translocation of the newly synthesized IRAP protein.     

Endocytosis and sorting of glycosphingolipids in sphingolipid storage disease

Recent studies on the endocytic itinerary of glycosphingolipids (GSLs) in sphingolipid storage disease (SLSD) fibroblasts have yielded new insights into the mechanisms underlying the endocytosis and intracellular sorting of lipids in normal and disease cells. Here we highlight new data on clathrin-independent endocytosis of GSLs, the involvement of sphingolipid–cholesterol interactions in perturbation of endocytic trafficking, and potential roles for rab proteins in regulation of GSL transport in SLSDs.     

Influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis

Influenza virus has been described to enter host cells via clathrin-mediated endocytosis. However, it has also been suggested that other endocytic routes may provide additional entry pathways. Here we show that influenza virus may enter and infect HeLa cells that are unable to take up ligands by clathrin-mediated endocytosis. By overexpressing a dominant-negative form of the Eps15 protein to inhibit clathrin-mediated endocytosis, we demonstrate that while transferrin uptake and Semliki Forest virus infection were prevented, influenza virus could enter and infect cells expressing Eps15Delta95/295. This finding is supported by the successful infection of cells with influenza virus in the presence of chemical treatments that block endocytosis, namely, chlorpromazine and potassium depletion. We show also that influenza virus may infect cells incapable of uptake by caveolae. Treatment with the inhibitors nystatin, methyl-beta-cyclodextrin, and genistein, as well as transfection of cells with dominant-negative caveolin-1, had no effect on influenza virus infection. By combining inhibitory methods to block both clathrin-mediated endocytosis and uptake by caveolae in the same cell, we demonstrate that influenza virus may infect cells by an additional non-clathrin-dependent, non-caveola-dependent endocytic pathway. We believe this to be the first conclusive analysis of virus entry via such a non-clathrin-dependent pathway, in addition to the traditional clathrin-dependent route.     

Oxidative stress disrupts internalization and endocytic trafficking of transferrin in a human malignant keratinocyte line

Oxidative stress is involved in epidermal cell pathology. One potential mechanism for this toxicity that has previously not been explored in epidermal cells involves modulation of endocytic trafficking and the implications that such modulation can have for altered cell function. The effects of oxidative stress on endocytic trafficking are not well understood, particularly relating to how general or cell-type specific such effects may be. With induction of oxidative stress by hydrogen peroxide, for example, both impaired and enhanced cell-surface binding and endocytic trafficking have been reported for transferrin (Tf), a circulatory iron-carrier protein. The objective of the current study was to characterize the effect of oxidative stress on internalization and endocytic trafficking of Tf in an epidermoid cell line (A431). Evidence is presented for a significant dose-dependent impairment of cellular Tf internalization after treatment with hydrogen peroxide over a wide range of concentrations from 0.06 to 5.8 mM. Scatchard analysis of binding revealed that peroxide treatments resulted in a large decrease, more than fourfold, in the number of cell-surace Tf-binding sites (Bmax) but little change in the dissociation constant (Kd). With respect to endocytic trafficking of Tf, evidence is presented that transport of internalized transferrin back out of the cell (i.e., Tf recycling) is significantly impaired as a result of oxidative stress at all the peroxide concentrations tested. The oxidative stress-dependent changes in endocytic trafficking in these malignant human keratinocytes are compared with those reported for other cell types.     

Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles

The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-beta-cyclodextrin (MbetaCD) to selectively extract cholesterol from the plasma membrane. MbetaCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MbetaCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MbetaCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MbetaCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.     

The Effect of Substrate Elasticity and Actomyosin Contractility on Different Forms of Endocytosis

Substrate mechanical properties have emerged as potent determinants of cell functions and fate. We here tested the hypothesis that different forms of endocytosis are regulated by the elasticity of the synthetic hydrogels cells are cultured on. Towards this objective, we quantified cell-associated fluorescence of the established endocytosis markers transferrin (Tf) and cholera toxin subunit B (CTb) using a flow-cytometry based protocol, and imaged marker internalization using microscopy techniques. Our results demonstrated that clathrin-mediated endocytosis of Tf following a 10-minute incubation with a fibroblast cell line was lower on the softer substrates studied (5 kPa) compared to those with elasticities of 40 and 85 kPa. This effect was cancelled after 1-hour incubation revealing that intracellular accumulation of Tf at this time point did not depend on substrate elasticity. Lipid-raft mediated endocytosis of CTb, on the other hand, was not affected by substrate elasticity in the studied range of time and substrate elasticity. The use of pharmacologic contractility inhibitors revealed inhibition of endocytosis for both Tf and CTb after a 10-minute incubation and a dissimilar effect after 1 hour depending on the inhibitor type. Further, the internalization of fluorescent NPs, used as model drug delivery systems, showed a dependence on substrate elasticity, while transfection efficiency was unaffected by it. Finally, an independence on substrate elasticity of Tf and CTb association with HeLa cells indicated that there are cell-type differences in this respect. Overall, our results suggest that clathrin-mediated but not lipid-raft mediated endocytosis is potentially influenced by substrate mechanics at the cellular level, while intracellular trafficking and accumulation show a more complex dependence. Our findings are discussed in the context of previous work on how substrate mechanics affect the fundamental process of endocytosis and highlight important considerations for future studies.     

Truncated brush border myosin I affects membrane traffic in polarized epithelial cells

We investigate, in this study, the potential involvement of an acto-myosin-driven mechanism in endocytosis of polarized cells. We observed that depolymerization of actin filaments using latrunculin A decreases the rate of transferrin recycling to the basolateral plasma membrane of Caco-2 cells, and increases its delivery to the apical plasma membrane. To analyze whether a myosin was involved in endocytosis, we produced, in this polarized cell line, truncated, non-functional, brush border, myosin I proteins (BBMI) that we have previously demonstrated to have a dominant negative effect on endocytosis of unpolarized cells. These non-functional proteins affect the rate of transferrin recycling and the rate of transepithelial transport of dipeptidyl-peptidase IV from the basolateral plasma membrane to the apical plasma membrane. They modify the distribution of internalized endocytic tracers in apical multivesicular endosomes that are accessible to fluid phase tracers internalized from apical and basolateral plasma membrane domains. Altogether, these observations suggest that an acto-myosin-driven mechanism is involved in the trafficking of basolaterally internalized molecules to the apical plasma membrane.     

Effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of Hep 2 cells by picornaviruses

Potassium depletion after a brief exposure of the cells to hypotonic medium was used to inhibit endocytosis from coated pits in Hep 2 cells. After such treatment the endocytic uptake of transferrin was arrested, and electron microscopy revealed that virtually no coated pits were present at the cell surface, while smooth (uncoated) pits were abundant. Under the same conditions the cells were strongly protected against poliovirus, while the cytopathogenic effect of human rhinovirus type 2, HRV 2, was increased. The cytopathogenic effect of encephalomyocarditis (EMC) virus was only slightly affected. Potassium depletion without hypotonic shock reduced the endocytic uptake of transferrin 2-3-fold and the number of coated pits at the cell surface about 3-fold. Furthermore, the cells were not protected against poliovirus after such treatment. The data indicate that the productive uptake of poliovirus occurs by receptor-mediated endocytosis from coated pits, while the productive uptake of the other two picornaviruses may occur by another endocytic pathway. In order to efficiently arrest endocytosis from coated pits in these cells, hypotonic shock seems to be a critical component of the potassium depletion protocol.     

The PIKfyve inhibitor YM201636 blocks the continuous recycling of the tight junction proteins claudin-1 and claudin-2 in MDCK cells

Tight junctions mediate the intercellular diffusion barrier found in epithelial tissues but they are not static complexes; instead there is rapid movement of individual proteins within the junctions. In addition some tight junction proteins are continuously being endocytosed and recycled back to the plasma membrane. Understanding the dynamic behaviour of tight junctions is important as they are altered in a range of pathological conditions including cancer and inflammatory bowel disease. In this study we investigate the effect of treating epithelial cells with a small molecule inhibitor (YM201636) of the lipid kinase PIKfyve, a protein which is involved in endocytic trafficking. We show that MDCK cells treated with YM201636 accumulate the tight junction protein claudin-1 intracellularly. In contrast YM201636 did not alter the localization of other junction proteins including ZO-1, occludin and E-cadherin. A biochemical trafficking assay was used to show that YM201636 inhibited the endocytic recycling of claudin-1, providing an explanation for the intracellular accumulation. Claudin-2 was also found to constantly recycle in confluent MDCK cells and treatment with YM201636 blocked this recycling and caused accumulation of intracellular claudin-2. However, claudin-4 showed negligible endocytosis and no detectable intracellular accumulation occurred following treatment with YM201636, suggesting that not all claudins show the same rate of endocytic trafficking. Finally, we show that, consistent with the defects in claudin trafficking, incubation with YM201636 delayed formation of the epithelial permeability barrier. Therefore, YM201636 treatment blocks the continuous recycling of claudin-1/claudin-2 and delays epithelial barrier formation.     

Selective modulation of the endocytic uptake of ricin and fluid phase markers without alteration in transferrin endocytosis

Cytochalasin D was found to reduce the endocytosis of ricin and the fluid phase markers [14C]sucrose and Lucifer Yellow in Vero cells without reducing the uptake of transferrin. The number of coated pits at the plasma membrane was not affected by the treatment. Cytochalasin D also reduced the endocytosis of ricin in cells where uptake of transferrin from coated pits was blocked by low cytosolic pH. Colchicine had a similar effect as cytochalasin D. Both drugs inhibited the exocytosis of ricin from the cells, and they reduced the rate by which ricin intoxicated the cells. Cytochalasin D had essentially no effect on the ability of the cells to bind transferrin, whereas colchicine reduced the binding to some extent. Epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) increased the endocytic uptake of ricin in A431 cells both under normal culture conditions and when the coated pit/coated vesicle pathway was blocked by acidification of the cytosol. In contrast, EGF and TPA had no stimulatory effect on the uptake of transferrin at normal cytoplasmic pH, and they did not abolish the ability of low cytoplasmic pH to inhibit endocytic uptake of transferrin. The results indicate that cytochalasin D and colchicine selectively inhibit endocytic uptake from non-clathrin-coated areas of the cell membrane whereas EGF and TPA stimulate it. The data support the view that there are different endocytic mechanisms, and they indicate that at least in some cell types the non-clathrin-coated endocytosis can be modulated.     

Infectious bursal disease virus uptake involves macropinocytosis and trafficking to early endosomes in a Rab5‐dependent manner

Infectious bursal disease virus (IBDV) internalization is sparsely known in terms of molecular components of the pathway involved. To describe the cell biological features of IBDV endocytosis, we employed perturbants of endocytic pathways such as pharmacological inhibitors and overexpression of dominant‐negative mutants. Internalization analysis was performed quantifying infected cells by immunofluorescence and Western blot detection of the viral protein VP3 at 12 h post‐infection reinforced by the analysis of the capsid protein VP2 localization after virus uptake at 1 h post‐infection. We compared IBDV infection to the internalization of well‐established ligands with defined endocytic pathways: transferrin, cholera‐toxin subunit B and dextran. To describe virus endocytosis at the morphological level, we performed ultrastructural studies of viral internalization kinetics in control and actin dynamics‐blocked cells. Our results indicate that IBDV endocytic internalization was clathrin‐ and dynamin‐independent, and that IBDV uses macropinocytosis as the primary entry mechanism. After uptake, virus traffics to early endosomes and requires exposure to the low endocytic pH as well as a functional endocytic pathway to complete its replication cycle. Moreover, our results indicate that the GTPase Rab5 is crucial for IBDV entry supporting the participation of the early endosomal pathway in IBDV internalization and infection of susceptible cells.     

Galectin-3 Guides Intracellular Trafficking of Some Human Serotransferrin Glycoforms

Transferrin internalization via clathrin-mediated endocytosis and subsequent recycling after iron delivery has been extensively studied. Here we demonstrate a previously unrecognized parameter regulating this recycling, the binding of galectin-3 to particular glycoforms of transferrin. Two fractions of transferrin, separated by affinity chromatography based on their binding or not to galectin-3, are targeted to kinetically different endocytic pathways in HFL-1 cells expressing galectin-3 but not in SKBR3 cells lacking galectin-3; the SKBR3 cells, however, can acquire the ability to target these transferrin glycoforms differently after preloading with exogenously added galectin-3. In all, this study provides the first evidence of a functional role for transferrin glycans, in intracellular trafficking after uptake. Moreover, the galectin-3-bound glycoform increased in cancer, suggesting a pathophysiological regulation. These are novel aspects of transferrin cell biology, which has previously considered only a degree of iron loading, but not other forms of heterogeneity.     

Role of protein kinase C betaII in influenza virus entry via late endosomes

Many viruses take advantage of receptor-mediated endocytosis in order to enter target cells. We have utilized influenza virus and Semliki Forest virus (SFV) to define a role for protein kinase C betaII (PKCbetaII) in endocytic trafficking. We show that specific PKC inhibitors prevent influenza virus infection, suggesting a role for classical isoforms of PKC. We also examined virus entry in cells overexpressing dominant-negative forms of PKCalpha and -beta. Cells expressing a phosphorylation-deficient form of PKCbetaII (T500V), but not an equivalent mutant form of PKCalpha, inhibited successful influenza virus entry-with the virus accumulating in late endosomes. SFV, however, believed to enter cells from the early endosome, was unaffected by PKCbetaII T500V expression. We also examined the trafficking of two cellular ligands, transferrin and epidermal growth factor (EGF). PKCbetaII T500V expression specifically blocked EGF receptor trafficking and degradation, without affecting transferrin receptor recycling. As with influenza virus, in PKCbetaII kinase-dead cells, EGF receptor was trapped in a late endosome compartment. Our findings suggest that PKCbetaII is an important regulator of a late endosomal sorting event needed for influenza virus entry and infection.