Ultrastructural Analysis of Nanogold-labeled Endocytic Compartments of Yeast Saccharomyces cerevisiae Using a Cryosectioning Procedure

Yeast Saccharomyces cerevisiae has been a valuable model organism for the study of the endosomal system of eukaryotic cells. Morphological analyses, however, have been limited because of the lack of specific protein markers and of procedures that lead to a satisfactory ultrastructural resolution. We have recently developed an immunoelectron microscopy (IEM) protocol adapted from the Tokuyasu method to prepare cryosections from mildly fixed yeast. This novel approach allows excellent cell preservation and a unique resolution of the yeast morphology. Here, we present a protocol that combines this procedure with the specific labeling of the various endosomal compartments with positively charged Nanogold. In particular, we show that this new protocol generates excellent results when applied for the examination of early and late endosomes, and of mutants with an endosomal trafficking defect. Importantly, this method is compatible with immunogold labeling of protein markers, and it is consequently appropriate for localization studies of both resident and cargo proteins. This new IEM protocol will be a valuable tool for the large community of scientists using yeast as a model system to investigate the membrane transport and the biogenesis of the endosomal system. (J Histochem Cytochem 57:801–809, 2009)     

Reorganization of the Endosomal System in Salmonella-Infected Cells: The Ultrastructure of Salmonella-Induced Tubular Compartments

During the intracellular life of Salmonella enterica, a unique membrane-bound compartment termed Salmonella-containing vacuole, or SCV, is formed. By means of translocated effector proteins, intracellular Salmonella also induce the formation of extensive, highly dynamic membrane tubules termed Salmonella-induced filaments or SIF. Here we report the first detailed ultrastructural analyses of the SCV and SIF by electron microscopy (EM), EM tomography and live cell correlative light and electron microscopy (CLEM). We found that a subset of SIF is composed of double membranes that enclose portions of host cell cytosol and cytoskeletal filaments within its inner lumen. Despite some morphological similarities, we found that the formation of SIF double membranes is independent from autophagy and requires the function of the effector proteins SseF and SseG. The lumen of SIF network is accessible to various types of endocytosed material and our CLEM analysis of double membrane SIF demonstrated that fluid phase markers accumulate only between the inner and outer membrane of these structures, a space continual with endosomal lumen. Our work reveals how manipulation of the endosomal membrane system by an intracellular pathogen results in a unique tubular membrane compartmentalization of the host cell, generating a shielded niche permissive for intracellular proliferation of Salmonella.     

Endocytosis in flight-stimulated adipokinetic cells ofLocusta migratoria

An ultrastructural morphometric study was made of the influence of flight activity on endocytosis in the adipokinetic cells ofLocusta migratoria. The endocytotic pathway was revealed by the endocytotic tracer horseradish peroxidase. Endocytosis appeared to be stimulated by flight, as indicated by an increase in the number of tracer-containing endocytotic pits and various intracellular endocytotic and lysosomal organelles. This stimulatory effect continued for at least 10 min after cessation of flight. An increase in the numbers of both tracer-labelled endocytotic pits and endosomal tubules was detected in the cell bodies of flight-stimulated adipokinetic cells. Endosomal tubules are supposed to be involved in recycling membrane material to the plasma membrane soon after it has been endocytosed. It is concluded that the increase in endocytosis in the flight-stimulated cell bodies serves to enlarge the uptake of nutritional and/or regulatory substances. An increase in number of tracer-labelled endocytotic pits was also observed in the cell processes of flight-stimulated adipokinetic cells, which was, however, not accompanied by an increase in number of labelled endosomal tubules, the latter being practically absent in the processes. This indicates that the increase in endocytotic activity in the cell processes is a form of adaptive endocytosis that compensates for membrane material added to the plasma membrane during flight-induced exocytotic release of adipokinetic hormones.     

Molecular assemblies and membrane domains in multivesicular endosome dynamics

Along the degradation pathway, endosomes exhibit a characteristic multivesicular organization, resulting from the budding of vesicles into the endosomal lumen. After endocytosis and transport to early endosomes, activated signaling receptors are incorporated into these intralumenal vesicles through the action of the ESCRT machinery, a process that contributes to terminate signaling. Then, the vesicles and their protein cargo are further transported towards lysosomes for degradation. Evidence also shows that intralumenal vesicles can undergo “back-fusion” with the late endosome limiting membrane, a route exploited by some pathogens and presumably followed by proteins and lipids that need to be recycled from within the endosomal lumen. This process depends on the late endosomal lipid lysobisphosphatidic acid and its putative effector Alix/AIP1, and is presumably coupled to the invagination of the endosomal limiting membrane at the molecular level via ESCRT proteins. In this review, we discuss the intra-endosomal transport routes in mammalian cells, and in particular the different mechanisms involved in membrane invagination, vesicle formation and fusion in a space inaccessible to proteins known to control intracellular membrane traffic.     

The Sec1/Munc18 Protein Vps45 Regulates Cellular Levels of Its SNARE Binding Partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic.     

Ultrastructural analysis of ESCRT proteins suggests a role for endosome-associated tubular-vesicular membranes in ESCRT function

The endosomal sorting complex required for transport (ESCRT) is thought to support the formation of intralumenal vesicles of multivesicular bodies (MVBs). The ESCRT is also required for the budding of HIV and has been proposed to be recruited to the HIV-budding site, the plasma membrane of T cells and MVBs in macrophages. Despite increasing data on the function of ESCRT, the ultrastructural localization of its components has not been determined. We therefore localized four proteins of the ESCRT machinery in human T cells and macrophages by quantitative electron microscopy. All the proteins were found throughout the endocytic pathway, including the plasma membrane, with only around 10 and 3% of the total labeling in the cytoplasm and on the MVBs, respectively. The majority of the labeling (45%) was unexpectedly found on tubular-vesicular endosomal membranes rather than on endosomes themselves. The ESCRT labeling was twice as concentrated on early and late endosomes/lysosomes in macrophages compared with that in T cells, where it was twice more abundant at the plasma membrane. The ESCRT proteins were not redistributed on HIV infection, suggesting that the amount of ESCRT proteins located at the budding site suffices for HIV release. These results represent the first systematic ultrastructural localization of ESCRT and provide insights into its role in uninfected and HIV-infected cells.     

The novel endosomal membrane protein Ema interacts with the class C Vps–HOPS complex to promote endosomal maturation

Endosomal maturation is critical for accurate and efficient cargo transport through endosomal compartments. Here we identify a mutation of the novel Drosophila gene, ema (endosomal maturation defective) in a screen for abnormal synaptic overgrowth and defective protein trafficking. Ema is an endosomal membrane protein required for trafficking of fluid-phase and receptor-mediated endocytic cargos. In the ema mutant, enlarged endosomal compartments accumulate as endosomal maturation fails, with early and late endosomes unable to progress into mature degradative late endosomes and lysosomes. Defective endosomal down-regulation of BMP signaling is responsible for the abnormal synaptic overgrowth. Ema binds to and genetically interacts with Vps16A, a component of the class C Vps–HOPS complex that promotes endosomal maturation. The human orthologue of ema, Clec16A, is a candidate susceptibility locus for autoimmune disorders, and its expression rescues the Drosophila mutant demonstrating conserved function. Characterizing this novel gene family identifies a new component of the endosomal pathway and provides insights into class C Vps–HOPS complex function.     

Ultrastructural Changes Caused by Snf7 RNAi in Larval Enterocytes of Western Corn Rootworm (Diabrotica virgifera virgifera Le Conte)

The high sensitivity to oral RNA interference (RNAi) of western corn rootworm (WCR, Diabrotica virgifera virgifera Le Conte) provides a novel tool for pest control. Previous studies have shown that RNAi of DvSnf7, an essential cellular component of endosomal sorting complex required for transport (ESCRT), caused deficiencies in protein de-ubiquitination and autophagy, leading to WCR death. Here we investigated the detailed mechanism leading to larval death by analyzing the ultrastructural changes in midgut enterocytes of WCR treated with double-stranded RNA (ds-DvSnf7). The progressive phases of pathological symptoms caused by DvSnf7-RNAi in enterocytes include: 1) the appearance of irregularly shaped macroautophagic complexes consisting of relatively large lysosomes and multi-lamellar bodies, indicative of failure in autolysosome formation; 2) cell sloughing and loss of apical microvilli, and eventually, 3) massive loss of cellular contents indicating loss of membrane integrity. These data suggest that the critical functions of Snf7 in insect midgut cells demonstrated by the ultrastructural changes in DvSnf7 larval enterocytes underlies the conserved essential function of the ESCRT pathway in autophagy and membrane stability in other organisms.     

Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2

During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747–1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion.     

Ultrastructural localization of Hsp-72 examined with a new polyclonal antibody raised against the truncated variable domain of the heat shock protein

In spite of the intensive search for the determination of the continuously widening physiological and pathological roles of different stress proteins, their ultrastructural localization at the electron microscopic (EM) level has hardly been examined. As it becomes increasingly evident that the function and physiological effectiveness of stress proteins are highly dependent on their spatial location and their associations with diverse regulator proteins, the demand for morphological studies which can identify their detailed distribution within the cells is evident. The reason for the practical lack of studies carried out at the EM level, lies in the shortage of reagents with suitable specificity and avidity necessary for this type of examination. To create such a reagent, a polyclonal antibody was raised using a recombinant truncated form of the inducible Hsp-72 protein. The antibody was extensively characterized, using different immunochemical methods to determine and verify its specificity, and then it was tried in ultrastructural examinations. Using the new antibody, it was possible to analyze the intracellular distribution of Hsp-72 with the immunogold technique. The localization of Hsp-72 was demonstrated directly at the ultrastructural level in the cytoplasm (especially at the cisterns of the RER), in the nucleus (mainly around the heterochromatic regions) and at both sides of the nuclear envelope close to the membrane pores. Apart from these localizations, Hsp-72 was found in several membrane bordered intracellular structures, which mainly belong to the endosomal-lysosomal system. We provide the first morphological verification of the appearance of Hsp-72 on the surface of the cells. Also novel is the indication, that the stress protein may recycle from the cell surface using a common route which includes coated pits and the endosomal system.     

Nef-induced alteration of the early/recycling endosomal compartment correlates with enhancement of HIV-1 infectivity

human immunodeficiency virus type 1 (HIV-1) Nef interacts with the clathrin-associated AP-1 and AP-3 adaptor complexes, stabilizing their association with endosomal membranes. These findings led us to hypothesize a general impact of this viral protein on the endosomal system. Here, we have shown that Nef specifically disturbs the morphology of the early/recycling compartment, inducing a redistribution of early endosomal markers and a shortening of the tubular recycling endosomal structures. Furthermore, Nef modulates the trafficking of the transferrin receptor (TfR), the prototypical recycling surface protein, indicating that it also disturbs the function of this compartment. Nef reduces the rate of recycling of TfR to the plasma membrane, causing TfR to accumulate in early endosomes and reducing its expression at the cell surface. These effects depend on the leucine-based motif of Nef, which is required for the membrane stabilization of AP-1 and AP-3 complexes. Since we show that this motif is also required for the full infectivity of HIV-1 virions, these results indicate that the positive influence of Nef on viral infectivity may be related to its general effects on early/recycling endosomal compartments.     

Regulation of Endosome Sorting by a Specific PP2A Isoform

The regulated sorting of proteins within the trans-Golgi network (TGN)/endosomal system is a key determinant of their biological activity in vivo. For example, the endoprotease furin activates of a wide range of proproteins in multiple compartments within the TGN/endosomal system. Phosphorylation of its cytosolic domain by casein kinase II (CKII) promotes the localization of furin to the TGN and early endosomes whereas dephosphorylation is required for efficient transport between these compartments (Jones, B.G., L. Thomas, S.S. Molloy, C.D. Thulin, M.D. Fry, K.A. Walsh, and G. Thomas. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:5869–5883). Here we show that phosphorylated furin molecules internalized from the cell surface are retained in a local cycling loop between early endosomes and the plasma membrane. This cycling loop requires the phosphorylation state-dependent furin-sorting protein PACS-1, and mirrors the trafficking pathway described recently for the TGN localization of furin (Wan, L., S.S. Molloy, L. Thomas, G. Liu, Y. Xiang, S.L. Ryback, and G. Thomas. 1998. Cell. 94:205–216). We also demonstrate a novel role for protein phosphatase 2A (PP2A) in regulating protein localization in the TGN/endosomal system. Using baculovirus recombinants expressing individual PP2A subunits, we show that the dephosphorylation of furin in vitro requires heterotrimeric phosphatase containing B family regulatory subunits. The importance of this PP2A isoform in directing the routing of furin from early endosomes to the TGN was established using SV-40 small t antigen as a diagnostic tool in vivo. The role of both CKII and PP2A in controlling multiple sorting steps in the TGN/endosomal system indicates that the distribution of itinerant membrane proteins may be acutely regulated via signal transduction pathways.     

Mechanisms Governing the Endosomal Membrane Recruitment of the Core Retromer in Arabidopsis

The retromer complex localizes to endosomal membranes and is involved in protein trafficking. In mammals, it is composed of a dimer of sorting nexins and of the core retromer consisting of vacuolar protein sorting (VPS)26, VPS29, and VPS35. Although homologs of these proteins have been identified in plants, how the plant retromer functions remains elusive. To better understand the role of VPS components in the assembly and function of the core retromer, we characterize here Arabidopsis vps26-null mutants. We show that impaired VPS26 function has a dramatic effect on VPS35 levels and causes severe phenotypic defects similar to those observed in vps29-null mutants. This implies that functions of plant VPS26, VPS29, and VPS35 are tightly linked. Then, by combining live-cell imaging with immunochemical and genetic approaches, we report that VPS35 alone is able to bind to endosomal membranes and plays an essential role in VPS26 and VPS29 membrane recruitment. We also show that the Arabidopsis Rab7 homolog RABG3f participates in the recruitment of the core retromer to the endosomal membrane by interacting with VPS35. Altogether our data provide original information on the molecular interactions that mediate assembly of the core retromer in plants.     

ESCRT components ISTL1 andLIP5 are required for tapetal function and pollen viability

Pollen wall assembly is crucial for pollen development and plant fertility. The durable biopolymer sporopollenin and the constituents of the tryphine coat are delivered to developing pollen grains by the highly coordinated secretory activity of the surrounding tapetal cells. The role of membrane trafficking in this process, however, is largely unknown. In this study, we used Arabidopsis thaliana to characterize the role of two late-acting endosomal sorting complex required for transport (ESCRT) components, ISTL1 and LIP5, in tapetal function. Plants lacking ISTL1 and LIP5 form pollen with aberrant exine patterns, leading to partial pollen lethality. We found that ISTL1 and LIP5 are required for exocytosis of plasma membrane and secreted proteins in the tapetal cells at the free microspore stage, contributing to pollen wall development and tryphine deposition. Whereas the ESCRT machinery is well known for its role in endosomal trafficking, the function of ISTL1 and LIP5 in exocytosis is not a typical ESCRT function. The istl1 lip5 double mutants also show reduced intralumenal vesicle concatenation in multivesicular endosomes in both tapetal cells and developing pollen grains as well as morphological defects in early endosomes/trans-Golgi networks, suggesting that late ESCRT components function in the early endosomal pathway and exocytosis.

Endosomal sorting complex required for transport proteins ISTL1 and LIP5 are required for exocytosis of both plasma membrane and secreted proteins in tapetal cells during microspore formation.     

Membrane Localization of β-Amyloid 1–42 in Lysosomes: A POSSIBLE MECHANISM FOR LYSOSOME LABILIZATION*

β-Amyloid peptide (Aβ42) is the core protein of amyloid plaque in Alzheimer disease. The intracellular accumulation of Aβ42 in the endosomal/lysosomal system has been under investigation for many years, but the direct link between Aβ42 accumulation and dysfunction of the endosomal/lysosomal system is still largely unknown. Here, we found that both in vitro and in vivo, a major portion of Aβ42 was tightly inserted into and a small portion peripherally associated with the lysosomal membrane, whereas its soluble portion was minimal. We also found that the Aβ42 molecules inserted into the membrane tended to form multiple oligomeric aggregates, whereas Aβ40 peptides formed only dimers. Neutralizing lysosomal pH in differentiated PC12 cells decreased the lysosomal membrane insertion of Aβ42 and moderated Aβ42-induced lysosomal labilization and cytotoxicity. Our findings, thus, suggest that the membrane-inserted portion of Aβ42 accumulated in lysosomes may destabilize the lysosomal membrane and induce neurotoxicity.     

The biogenesis and functions of exosomes

Exosomes are membrane vesicles with a diameter of 40–100 nm that are secreted by many cell types into the extracellular milieu. They correspond to the internal vesicles of an endosomal compartment, the multivesicular body and are released upon exocytic fusion of this organelle with the plasma membrane. Intracellularly, they are formed by inward budding of the endosomal membrane in a process that sequesters particular proteins and lipids. The unique composition of exosomes may confer specific functions on them upon secretion. Although their physiological role in vivo is far from being unraveled, it is apparent that they function in a multitude of processes, including intercellular communication during the immune response. Exosomes may have evolved early in the evolution of multicellular organisms and also seem to be important for tissue developmental processes.     

Defining the Range of Pathogens Susceptible to Ifitm3 Restriction Using a Knockout Mouse Model

The interferon-inducible transmembrane (IFITM) family of proteins has been shown to restrict a broad range of viruses in vitro and in vivo by halting progress through the late endosomal pathway. Further, single nucleotide polymorphisms (SNPs) in its sequence have been linked with risk of developing severe influenza virus infections in humans. The number of viruses restricted by this host protein has continued to grow since it was first demonstrated as playing an antiviral role; all of which enter cells via the endosomal pathway. We therefore sought to test the limits of antimicrobial restriction by Ifitm3 using a knockout mouse model. We showed that Ifitm3 does not impact on the restriction or pathogenesis of bacterial (Salmonella typhimurium, Citrobacter rodentium, Mycobacterium tuberculosis) or protozoan (Plasmodium berghei) pathogens, despite in vitro evidence. However, Ifitm3 is capable of restricting respiratory syncytial virus (RSV) in vivo either through directly restricting RSV cell infection, or by exerting a previously uncharacterised function controlling disease pathogenesis. This represents the first demonstration of a virus that enters directly through the plasma membrane, without the need for the endosomal pathway, being restricted by the IFITM family; therefore further defining the role of these antiviral proteins.     

The dark side of lysosome-related organelles: specialization of the endocytic pathway for melanosome biogenesis

Melanosomes are lysosome-related organelles within which melanin pigments are synthesized and stored in melanocytes and retinal pigment epithelial cells. Early ultrastructural studies of pigment cells revealed that melanosomes consist of a complex series of organelles; more recently, these structures have been correlated with cargo constituents. By studying the fate of melanosomal and endosomal cargo in melanocytic cells, the effects of disease-related mutations on melanosomal morphology, and the genes affected by these mutations, we are beginning to gain novel insights into the biogenesis of these complex organelles and their relationship to the endocytic pathway. These insights demonstrate how specialized cells integrate unique and ubiquitous molecular mechanisms in subverting the endosomal system to generate cell-type specific structures and their associated functions. Further dissection of the melanosomal system will likely shed light not only on the biogenesis of lysosome-related organelles but also on general aspects of vesicular transport in the endosomal system.     

Pkh1/2-dependent phosphorylation of Vps27 regulates ESCRT-I recruitment to endosomes

Multivesicular endosomes (MVBs) are major sorting platforms for membrane proteins and participate in plasma membrane protein turnover, vacuolar/lysosomal hydrolase delivery, and surface receptor signal attenuation. MVBs undergo unconventional inward budding, which results in the formation of intraluminal vesicles (ILVs). MVB cargo sorting and ILV formation are achieved by the concerted function of endosomal sorting complex required for transport (ESCRT)-0 to ESCRT-III. The ESCRT-0 subunit Vps27 is a key player in this pathway since it recruits the other complexes to endosomes. Here we show that the Pkh1/Phk2 kinases, two yeast orthologues of the 3-phosphoinositide–dependent kinase, phosphorylate directly Vps27 in vivo and in vitro. We identify the phosphorylation site as the serine 613 and demonstrate that this phosphorylation is required for proper Vps27 function. Indeed, in pkh-ts temperature-sensitive mutant cells and in cells expressing vps27^S613A, MVB sorting of the carboxypeptidase Cps1 and of the α-factor receptor Ste2 is affected and the Vps28–green fluorescent protein ESCRT-I subunit is mainly cytoplasmic. We propose that Vps27 phosphorylation by Pkh1/2 kinases regulates the coordinated cascade of ESCRT complex recruitment at the endosomal membrane.     

Altered GLUT4 trafficking in adipocytes in the absence of the GTPase Arfrp1

The GTPase ADP-ribosylation factor related protein 1 (ARFRP1) controls the recruitment of proteins such as golgin-245 to the trans-Golgi. ARFRP1 is highly expressed in adipose tissues in which the insulin-sensitive glucose transporter GLUT4 is processed through the Golgi to a specialized endosomal compartment, the insulin-responsive storage compartment from which it is translocated to the plasma membrane in response to a stimulation of cells by insulin. In order to examine the role of ARFRP1 for GLUT4 targeting, subcellular distribution of GLUT4 was investigated in adipose tissue specific Arfrp1 knockout (Arfrp1^ad−/−) mice. Immunohistochemical and ultrastructural studies of brown adipocytes demonstrated an abnormal trans-Golgi in Arfrp1^ad−/− adipocytes. In addition, in Arfrp1^ad−/− adipocytes GLUT4 protein accumulated at the plasma membrane rather than being sequestered in an intracellular compartment. A similar missorting of GLUT4 was produced by siRNA-mediated knockdown of Arfrp1 in 3T3-L1 adipocytes which was associated with significantly elevated uptake of deoxyglucose under basal conditions. Thus, Arfrp1 appears to be involved in sorting of GLUT4.