Characterization of rapid membrane internalization and recycling

Lipids and other membrane constituents recycle between the plasma membrane and intracellular endocytic compartments. In CHO cells, approximately half of the internalized C(6)-NBD-SM, a fluorescent lipid analogue widely used as a membrane maker, recycles via the endocytic recycling compartment with a t(12) of approximately 12 min (Mayor, S., Presley, J. F., and Maxfield, F. R. (1993) J. Cell Biol. 121, 1257-1269). Surprisingly, the rest returns to the plasma membrane very quickly. A detailed kinetic study presented in this paper indicates that after a brief internalization pulse, 42-62% of the internalized C(6)-NBD-SM returns to the plasma membrane with a t(12) of 1-2 min. Similar results are obtained using HEp2 and nonpolarized Madin-Darby canine kidney cells. Using FM dyes of different hydrophobicity, we show that rapid recycling involves passage through an endocytic organelle that was subsequently identified as the sorting endosome by co-localization with internalized transferrin and low density lipoprotein. These results imply that the membrane internalization rate is much higher than previously estimated, with a t(12) as short as 5-10 min. Rapid internalization and recycling would facilitate processes such as nutrient uptake and cholesterol efflux.     

Slowed receptor trafficking in mutant CHO lines of the end1 and end2 complementation groups

A mutant Chinese hamster ovary cell line with nonconditional kinetic defects in receptor internalization and recycling was isolated, based on selection for resistance to a transferrin-diphtheria toxin conjugate and screening for aberrant receptor trafficking. The 12-4 cell line internalizes transferrin at ～75% of the parental rate and recyles transferrin back to the cell surface at ～55% of the parental rate. Internalization of low density lipoprotein is also reduced to ～70% of the parental cell rate, demonstrating that the mutant phenotype affects the trafficking of multiple receptors. Characterization of somatic cell hybrids indicated that the 12-4 phenotype is recessive, and complementation analysis determined that the 12-4 cell line is a member of the End2 complementation group. End2 mutants have previously been described as defective in endosomal acidification but have not been known to be defective in receptor trafficking. We have found similar defects in another End2 mutant cell line, suggesting that slowed receptor trafficking is characteristic of End2 mutants. Interestingly, transferrin receptor recycling and internalization are also slowed in another complementation group of mutants, End1, that is also defective in endosomal acidification. This study demonstrates altered receptor trafficking in End1 and End2 cell lines, a novel aspect of the mutant phenotypes. These findings provide evidence, based on a cellular genetic approach, that proper endosome acidification is necessary for maintenance of normal receptor recycling. © 1994 Wiley-Liss, Inc.     

Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process

A central question in the endocytic process concerns the mechanism for sorting of recycling components (such as transferrin or low density lipoprotein receptors) from lysosomally directed components; membrane- associated molecules including receptors are generally directed towards the recycling pathway while the luminal content of sorting endosomes, consisting of the acid-released ligands, are lysosomally targeted. However, it is not known whether recycling membrane receptors follow bulk membrane flow or if these proteins are actively sorted from lysosomally directed material because of specific protein sequences and/or structural features. Using quantitative fluorescence microscopy we have determined the endocytic route and kinetics of traffic of the bulk carrier, membrane lipids, to address this issue directly. We show that N-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-epsilon-aminohexanoyl]- sphingosylphosphorylcholine (C6-NBD-SM) in endocytosed as bulk membrane, and it transits the endocytic system kinetically and morphologically identically to fluorescently labeled transferrin in a CHO cell line. With indistinguishable kinetics, the two labeled markers sort from lysosomally destined molecules in peripherally located sorting endosomes, accumulate in a peri-centriolar recycling compartment, and finally exit the cell. Other fluorescently labeled lipids, C6-NBD-phosphatidylcholine and galactosylceramide also traverse the same pathway. The constitutive nature of sorting of bulk membrane towards the recycling pathway and the lysosomal direction of fluid phase implies a geometric basis of sorting.     

Rab4 affects both recycling and degradative endosomal trafficking

The small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen-1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways.     

Rab11 regulates recycling through the pericentriolar recycling endosome

Small GTPases of the rab family are crucial elements of the machinery that controls membrane traffic. In the present study, we examined the distribution and function of rab11. Rab11 was shown by confocal immunofluorescence microscopy and EM to colocalize with internalized transferrin in the pericentriolar recycling compartment of CHO and BHK cells. Expression of rab11 mutants that are preferentially in the GTP- or GDP-bound state caused opposite effects on the distribution of transferrin-containing elements; rab11-GTP expression caused accumulation of labeled elements in the perinuclear area of the cell, whereas rab11-GDP caused a dispersion of the transferrin labeling. Functional studies showed that the early steps of uptake and recycling for transferrin were not affected by overexpression of rab11 proteins. However, recycling from the later recycling endosome was inhibited in cells overexpressing the rab11-GDP mutant. Rab5, which regulates early endocytic trafficking, acted before rab11 in the transferrin-recycling pathway as expression of rab5-GTP prevented transport to the rab11- positive recycling endosome. These results suggest a novel role for rab11 in controlling traffic through the recycling endosome.     

Rab11b is essential for recycling of transferrin to the plasma membrane

Members of the Rab family of small GTPases play important roles in membrane trafficking along the exocytic and endocytic pathways. The Rab11 subfamily consists of two highly conserved members, Rab11a and Rab11b. Rab11a has been localized both to the pericentriolar recycling endosome and to the trans-Golgi network and functions in recycling of transferrin. However, the localization and function of Rab11b are completely unknown. In this study green fluorescent protein (GFP)-tagged Rab11b was used to determine its subcellular localization. GFP-Rab11b colocalized with internalized transferrin, and using different mutants of Rab11b, the role of this protein in transferrin uptake and recycling was examined. Two of these mutants, Rab11b-Q/L (constitutively active) and Rab11b-S/N (constitutively inactive), strongly inhibited the recycling of transferrin. Interestingly, both of them had no effect on transferrin uptake. In contrast, the C-terminally altered mutant Rab11b-DeltaC, which cannot be prenylated and therefore cannot interact with membranes, did not interfere with wild-type Rab11b function. From these data we concluded that functional Rab11b is essential for the transport of internalized transferrin from the recycling compartment to the plasma membrane.     

Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts

We examined the metabolism and intracellular transport of the D-erythro and L-threo stereoisomers of a fluorescent analogue of sphingomyelin, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl])-sphingosylphosphorylcholine (C6-NBD-SM), in Chinese hamster ovary (CHO-K1) fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 10-15 min of being warmed to 37 degrees C, C6-NBD-SM was internalized from the plasma membrane to a perinuclear location that colocalized with the centriole and was distinct from the lysosomes and the Golgi apparatus. This perinuclear region was also labeled by internalized rhodamine-conjugated transferrin. C6-NBD-SM endocytosis was not inhibited when the microtubules were disrupted with nocodazole; rather, the fluorescent lipid was distributed in vesicles throughout the cell periphery instead of being internalized to the perinuclear region of the cell. The metabolism of C6-NBD-SM to other fluorescent sphingolipids at 37 degrees C and its effect on C6-NBD-SM transport was also examined. To study plasma membrane lipid recycling, C6-NBD-SM was first inserted into the plasma membrane of CHO-K1 cells and then allowed to be internalized by the cells at 37 degrees C. Any C6-NBD-SM remaining at the plasma membrane was then removed by incubation with nonfluorescent liposomes at 7 degrees C, leaving cells containing only internalized fluorescent lipid. The return of C6-NBD-SM to the plasma membrane from intracellular compartments upon further 37 degrees C incubation was then observed. The half-time for a complete round C6-NBD-SM recycling between the plasma membrane and intracellular compartments was approximately 40 min. Pretreatment of cells with either monensin or nocodazole did not inhibit C6-NBD-SM recycling.     

Over-expression of Rififylin, a new RING finger and FYVE-like domain-containing protein, inhibits recycling from the endocytic recycling compartment

Endocytosed membrane components are recycled to the cell surface either directly from early/sorting endosomes or after going through the endocytic recycling compartment (ERC). Studying recycling mechanisms is difficult, in part due to the fact that specific tools to inhibit this process are scarce. In this study, we have characterized a novel widely expressed protein, named Rififylin (Rffl) for RING Finger and FYVE-like domain-containing protein, that, when overexpressed in HeLa cells, induced the condensation of transferrin receptor-, Rab5-, and Rab11-positive recycling tubulovesicular membranes in the perinuclear region. Internalized transferrin was able to access these condensed endosomes but its exit from this compartment was delayed. Using deletion mutants, we show that the carboxy-terminal RING finger of Rffl is dispensable for its action. In contrast, the amino-terminal domain of Rffl, which shows similarities with the phosphatidylinositol-3-phosphate-binding FYVE finger, is critical for the recruitment of Rffl to recycling endocytic membranes and for the inhibition of recycling, albeit in a manner that is independent of PtdIns(3)-kinase activity. Rffl overexpression represents a novel means to inhibit recycling that will help to understand the mechanisms involved in recycling from the ERC to the plasma membrane.     

Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts

We examined the metabolism and intracellular transport of a fluorescent sphingomyelin analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl])- sphingosylphosphorylcholine (C6-NBD-SM), in both normal and Niemann-Pick, type A (NP-A) human skin fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 3 min of warming to 37 degrees C, both normal and NP-A fibroblasts had internalized C6-NBD-SM from the plasma membrane, resulting in a punctate pattern of intracellular fluorescence. Rates for C6-NBD-SM internalization and transport from intracellular compartments to the plasma membrane (recycling) were similar for normal and NP-A cells. With increasing time at 37 degrees C, internalized C6-NBD-SM accumulated in the lysosomes of NP-A fibroblasts, while normal fibroblasts showed increasing Golgi apparatus fluorescence with no observable lysosomal labeling. Since NP-A fibroblasts lack lysosomal (acid) sphingomyelinase (A-SMase), this result suggested that hydrolysis of C6-NBD-SM prevented its accumulation in the lysosomes of normal fibroblasts during its transport along the degradative pathway. We used the amount of C6-NBD-SM hydrolysis by A-SMase in normal cells as a measure of C6-NBD-SM transported from the cell surface to the lysosomes. After a lag period, C6-NBD-SM was delivered to the lysosomes at a rate of approximately 8%/h. This rate was approximately 18-19 fold slower than the rate of C6-NBD-SM recycling from intracellular compartments to the plasma membrane. Thus, small amounts of C6-NBD-SM were transported along the degradative pathway, while most endocytosed C6-NBD-SM was sorted for transport along the plasma membrane recycling pathway.     

Rab11 in recycling endosomes regulates the sorting and basolateral transport of E-cadherin

E-cadherin plays an essential role in cell polarity and cell-cell adhesion; however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. We first traced the post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, DeltaS1-E-cadherin, colocalized with Rab11 and fused with the recycling endosome before diverging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical DeltaS1-E-cadherin was unaffected. We thus demonstrate a novel pathway for Rab11 dependent, dileucine-mediated, mu1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion.     

Reconstitution of recycling from the phagosomal compartment in streptolysin O-permeabilized macrophages: role of Rab11

By phagocytosis, macrophages engulf large particles, microorganisms and senescent cells in vesicles called phagosomes. Many internalized proteins rapidly shuttle back to the plasma membrane following phagosome biogenesis. Here, we report a new approach to the study of recycling from the phagosomal compartment: streptolysin O- (SLO) permeabilized macrophages. In this semi-intact cell system, energy and cytosol are required to efficiently reconstitute recycling transport. Addition of GDPbetaS strongly inhibits this transport step, suggesting that a GTP-binding protein modulates the dynamics of cargo exit from the phagosomal compartment. GTPases of the Rab family control vesicular trafficking, and Rab11 is involved in transferrin receptor recycling. To unravel the role of Rab11 in the phagocytic pathway, we added recombinant proteins to SLO-permeabilized macrophages. Rab11:S25N, a negative mutant, strongly diminishes the release of recycled proteins from phagosomes. In contrast, wild type Rab11 and its positive mutant (Rab11:Q70L) favor this vesicular transport event. Using biochemical and morphological assays, we confirm that overexpression of Rab11:S25N substantially decreases recycling from phagosomes in intact cells. These findings show the requirement of a functional Rab11 for the retrieval to the plasma membrane of phagosomal content. SLO-permeabilized macrophages likely constitute a useful tool to identify new molecules involved in regulating transport along the phagocytic pathway.     

GPI anchoring leads to sphingolipid-dependent retention of endocytosed proteins in the recycling endosomal compartment

Glycosylphosphatidylinositol (GPI) anchoring is important for the function of several proteins in the context of their membrane trafficking pathways. We have shown previously that endocytosed GPI-anchored proteins (GPI-APs) are recycled to the plasma membrane three times more slowly than other membrane components. Recently, we found that GPI-APs are delivered to endocytic organelles, devoid of markers of the clathrin-mediated pathway, prior to their delivery to a common recycling endosomal compartment (REC). Here we show that the rate-limiting step in the recycling of GPI-APs is their slow exit from the REC; replacement of the GPI anchor with a transmembrane protein sequence abolishes retention in this compartment. Depletion of endogenous sphingolipid levels using sphingolipid synthesis inhibitors or in a sphingolipid-synthesis mutant cell line specifically enhances the rate of endocytic recycling of GPI-APs to that of other membrane components. We have shown previously that endocytic retention of GPI-APs is also relieved by cholesterol depletion. These findings strongly suggest that functional retention of GPI-APs in the REC occurs via their association with sphingolipid and cholesterol-enriched sorting platforms or 'rafts'.     

Rab15 effector protein: a novel protein for receptor recycling from the endocytic recycling compartment

Sorting endosomes and the endocytic recycling compartment are critical intracellular stores for the rapid recycling of internalized membrane receptors to the cell surface in multiple cell types. However, the molecular mechanisms distinguishing fast receptor recycling from sorting endosomes and slow receptor recycling from the endocytic recycling compartment remain poorly understood. We previously reported that Rab15 differentially regulates transferrin receptor trafficking through sorting endosomes and the endocytic recycling compartment, suggesting a role for distinct Rab15-effector interactions at these endocytic compartments. In this study, we identified the novel protein Rab15 effector protein (REP15) as a binding partner for Rab15-GTP. REP15 is compartment specific, colocalizing with Rab15 and Rab11 on the endocytic recycling compartment but not with Rab15, Rab4, or early endosome antigen 1 on sorting endosomes. REP15 interacts directly with Rab15-GTP but not with Rab5 or Rab11. Consistent with its localization, REP15 overexpression and small interfering RNA-mediated depletion inhibited transferrin receptor recycling from the endocytic recycling compartment, without affecting receptor entry into or recycling from sorting endosomes. Our data identify REP15 as a compartment-specific protein for receptor recycling from the endocytic recycling compartment, highlighting that the rapid and slow modes of transferrin receptor recycling are mechanistically distinct pathways.     

Rme-1 regulates the recycling of the cystic fibrosis transmembrane conductance regulator

Endocytic motifs in the carboxyl terminus of cystic fibrosis transmembrane conductance regulator (CFTR) direct internalization from the plasma membrane by clathrin-mediated endocytosis. However, the fate of such internalized CFTR has remained unknown. Internalized membrane proteins can be either targeted for degradation or recycled back to the plasma membrane. Using cell surface biotinylation and antibody uptake studies, we show that CFTR undergoes constitutive endocytosis and recycling back to the plasma membrane. Expression of dominant negative Rme-1 (a protein that regulates exit from the endosomal recycling compartment) in CFTR-expressing cells results in the expansion of recycling compartments. Transferrin, a marker for the endosomal recycling compartment, and CFTR accumulate in these enlarged recycling endosomes. Such accumulation leads to a loss of cell surface CFTR because it is prevented from being recycled back to the cell surface. In contrast, traffic of the low-density lipoprotein (LDL) is unaffected by the expression of dominant negative Rme-1. In addition, chimeras containing the extracellular domain of the transferrin receptor and the carboxyl terminal tail of CFTR also enter Rme-1-regulated recycling compartments and accumulate in these compartments containing dominant negative Rme-1, suggesting that in addition to endocytic signals, the carboxyl terminal tail of CFTR also contains intracellular traffic information.     

Evidence for nonvectorial, retrograde transferrin trafficking in the early endosomes of HEp2 cells

We have previously characterized the trafficking of transferrin (Tf) through HEp2 human carcinoma cells (Ghosh, R. N., D. L. Gelman, and F. R. Maxfield, 1994. J. Cell Sci. 107:2177-2189). Early endosomes in these cells are comprised of both sorting endosomes and recycling compartments, which are distinct separate compartments. Endocytosed Tf initially appears in punctate sorting endosomes that also contain recently endocytosed LDL. After short loading pulses, Tf rapidly sorts from LDL with first-order kinetics (t1/2 approximately 2.5 min), and it enters the recycling compartment before leaving the cell (t1/2 approximately 7 min). Here, we report a second, slower rate for Tf to leave sorting endosomes after HEp2 cells were labeled to steady state with fluorescein Tf instead of the brief pulse used previously. We determined this rate using digital image analysis to measure the Tf content of sorting endosomes that also contained LDL. With an 11-min chase, the Tf in sorting endosomes was 24% of steady-state value. This was in excess of the amount expected (5% of steady state) from the rate of Tf exit after short filling pulses. The excess could not be accounted for by reinternalization of recycled cell surface Tf, implying that either some Tf was retained in sorting endosomes, or that Tf was delivered back to the sorting endosomes from the recycling compartment. The former is unlikely since nearly all sorting endosomes contain detectable Tf after an 11-min chase, even though more than one third of the sorting endosomes were formed during the chase time. Furthermore, while observing living cells by confocal microscopy, we saw vesicle movements that appeared to be fluorescent Tf returning from recycling compartments to sorting endosomes. The slow rate of exit after steady-state labeling was similar to the Tf exit rate from the cell, suggesting an equilibration of Tf throughout the early endosomal system by this retrograde pathway. This retrograde traffic may be important for delivering molecules from the recycling compartment, which is a long-lived organelle, to sorting endosomes, which are transient.     

Proteomics identification of sorting nexin 27 as a diacylglycerol kinase zeta-associated protein: new diacylglycerol kinase roles in endocytic recycling

Diacylglycerol kinase zeta is a member of the diacylglycerol kinase family of enzymes, which generate phosphatidic acid through diacylglycerol phosphorylation. In addition to the catalytic and cysteine-rich domains found in all diacylglycerol kinases, diacylglycerol kinase zeta has a MARCKS domain as well as a C-terminal region containing four ankyrin repeats and a PDZ-binding motif. Previous reports demonstrated that diacylglycerol kinase zeta interaction with several proteins is an important mechanism for modulating the localization and activity of this enzyme. Here we used a proteomics approach to search for novel diacylglycerol kinase zeta-interacting proteins and identified sorting nexin 27 (SNX27), a recently described member of a protein family involved in intracellular trafficking, which has a PDZ domain in addition to the phox homology domain characteristic of SNX proteins. Co-immunoprecipitation studies and two-hybrid analysis confirmed physical, PDZ-dependent association between SNX27 and diacylglycerol kinase zeta. Because diacylglycerol kinase zeta is expressed abundantly in T lymphocytes, we characterized SNX27 expression and subcellular localization in these cells. SNX27 co-localized with transferrin receptor-positive vesicles, pointing to its participation in T cell endocytic recycling. Expression of deletion mutants revealed that in addition to the phox homology domain the SNX27 PDZ domain contributed to vesicle localization of this protein, suggesting that interaction with diacylglycerol kinase zeta regulates SNX27 localization. Analysis of cells with RNA interference-mediated knockdown of diacylglycerol kinase zeta showed accelerated transferrin receptor exit from the lymphocyte endocytic recycling compartment back to the plasma membrane, further confirming diacylglycerol kinase zeta-dependent control of vesicle trafficking. These data support a previously unreported role for diacylglycerol kinase zeta in the modulation of membrane trafficking, which may also help to define SNX27 function.     

The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes

The low-density lipoprotein receptor (LDLR) mediates the hepatic uptake of circulating low-density lipoproteins (LDLs), a process that modulates the development of atherosclerotic cardiovascular disease. We recently identified RAB10, encoding a small GTPase, as a positive regulator of LDL uptake in hepatocellular carcinoma cells (HuH7) in a genome-wide CRISPR screen, though the underlying molecular mechanism for this effect was unknown. We now report that RAB10 regulates hepatocyte LDL uptake by promoting the recycling of endocytosed LDLR from RAB11-positive endosomes to the plasma membrane. We also show that RAB10 similarly promotes the recycling of the transferrin receptor, which binds the transferrin protein that mediates the transport of iron in the blood, albeit from a distinct RAB4-positive compartment. Taken together, our findings suggest a model in which RAB10 regulates LDL and transferrin uptake by promoting both slow and rapid recycling routes for their respective receptor proteins.     

Isolation of a temperature-sensitive variant Chinese hamster ovary cell line with a morphologically altered endocytic recycling compartment

We have enriched a mutagenized population of Chinese hamster ovary (CHO) cells for those defective in endocytosis by selection for survival to treatment with transferrin (Tf)-ricin and Tf-diphtheria toxin conjugates. Surviving cells were screened with a fluorescently labeled Tf uptake assay to identify cells with mor-phologically aberrant endocytic phenotypes. One of the cell lines identified, B104-5, has a striking temperature-induced alteration in the morphology of its endocytic receptor recycling compartment. In parental cells the tightly clustered endocytic recycling compartment is located near the Golgi complex. In the mutant cells, following incubation at 40°C, this compartment appears fragmented and widely dispersed. Surprisingly, this alteration in the morphology of the recycling compartment has no effect on the kinetics of Tf internationalization and recycling. The wild-type endocytic compartment is closely aligned with the microtubule-organizing center and the Golgi apparatus, and like the Golgi, its clustered appearance is dependent upon intact microtubules. Although the disruption of the B104-5 receptor recycling compartment morphology can be phenocopied in wild-type cells by microtubule depolymerizing drugs, the microtubule cytoskeleton in B104-5 cells appears normal in immunofluorescent staining. B104-5 cells, unlike the parental cells, do not proliferate at 40°C. The mutation in B104-5 cells is recessive, as fusion with wild-type cells results in a reversion of the B104-5 phenotype. The finding that the morphology of the recycling compartment in CHO cells can be altered without affecting recycling of endocytosed Tf is consistent with the variety of recycling compartment morphologies observed among different cell lines. An interpretation of this result is that the lesion in B104-5 cells is in a gene that is involved in determining the endocytic compartment morphologies observed in different cell lines. © 1993 Wiley-Liss, Inc.     

Human Liver Cell Trafficking Mutants: Characterization and Whole Exome Sequencing

The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α’’. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.     

Phospholipase D and retromer promote recycling of TRPL ion channel via the endoplasmic reticulum

Plasma membrane protein trafficking is of fundamental importance for cell function and cell integrity of neurons and includes regulated protein recycling. In this work, we report a novel role of the endoplasmic reticulum (ER) for protein recycling as discovered in trafficking studies of the ion channel TRPL in photoreceptor cells of Drosophila. TRPL is located within the rhabdomeric membrane from where it is endocytosed upon light stimulation and stored in the cell body. Conventional immunohistochemistry as well as stimulated emission depletion super-resolution microscopy revealed TRPL storage at the ER after illumination, suggesting an unusual recycling route of TRPL. Our results also imply that both phospholipase D (PLD) and retromer complex are required for correct recycling of TRPL to the rhabdomeric membrane. Loss of PLD activity in PLD^3.1 mutants results in enhanced degradation of TRPL. In the retromer mutant vps35^MH20, TRPL is trapped in a Rab5-positive compartment. Evidenced by epistatic analysis in the double mutant PLD^3.1 vps35^MH20, PLD activity precedes retromer function. We propose a model in which PLD and retromer function play key roles in the transport of TRPL to an ER enriched compartment.