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1.
The HOPS/Class C Vps Complex Tethers High‐Curvature Membranes via a Direct Protein–Membrane Interaction 下载免费PDF全文
Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter‐membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sorting/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low‐curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly curved liposomes to Ypt7p‐bearing low‐curvature liposomes even when the high‐curvature liposomes are protein‐free. Phosphorylation of the curvature‐sensing amphipathic lipid‐packing sensor (ALPS) motif from the Vps41p HOPS subunit abrogates tethering of high‐curvature liposomes. A HOPS complex without its Vps39p subunit, which contains one of the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds high‐curvature liposomes and Ypt7p‐bearing low‐curvature liposomes. Thus, HOPS tethers highly curved membranes via a direct protein–membrane interaction. Such high‐curvature membranes are found at the sites of vacuole tethering and fusion. There, vacuole membranes bend sharply, generating large areas of vacuole‐vacuole contact. We propose that HOPS localizes via the Vps41p ALPS motif to these high‐curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane. 相似文献
2.
Rab24 interacts with the Rab7/Rab interacting lysosomal protein complex to regulate endosomal degradation 下载免费PDF全文
Celina Amaya Rodrigo D. Militello Sebastián D. Calligaris María I. Colombo 《Traffic (Copenhagen, Denmark)》2016,17(11):1181-1196
Endocytosis is a multistep process engaged in extracellular molecules internalization. Several proteins including the Rab GTPases family coordinate the endocytic pathway. The small GTPase Rab7 is present in late endosome (LE) compartments being a marker of endosome maturation. The Rab interacting lysosomal protein (RILP) is a downstream effector of Rab7 that recruits the functional dynein/dynactin motor complex to late compartments. In the present study, we have found Rab24 as a component of the endosome‐lysosome degradative pathway. Rab24 is an atypical protein of the Rab GTPase family, which has been attributed a function in vesicle trafficking and autophagosome maturation. Using a model of transiently expressed proteins in K562 cells, we found that Rab24 co‐localizes in vesicular structures labeled with Rab7 and LAMP1. Moreover, using a dominant negative mutant of Rab24 or a siRNA‐Rab24 we showed that the distribution of Rab7 in vesicles depends on a functional Rab24 to allow DQ‐BSA protein degradation. Additionally, by immunoprecipitation and pull down assays, we have demonstrated that Rab24 interacts with Rab7 and RILP. Interestingly, overexpression of the Vps41 subunit from the homotypic fusion and protein‐sorting (HOPS) complex hampered the co‐localization of Rab24 with RILP or with the lysosomal GTPase Arl8b, suggesting that Vps41 would affect the Rab24/RILP association. In summary, our data strongly support the hypothesis that Rab24 forms a complex with Rab7 and RILP on the membranes of late compartments. Our work provides new insights into the molecular function of Rab24 in the last steps of the endosomal degradative pathway. 相似文献
3.
Zaira Rizopoulos Giuseppe Balistreri Samuel Kilcher Caroline K. Martin Mohammedyaseen Syedbasha Ari Helenius Jason Mercer 《Traffic (Copenhagen, Denmark)》2015,16(8):814-831
The prototypic poxvirus, vaccinia virus (VACV), occurs in two infectious forms, mature virions (MVs) and extracellular virions (EVs). Both enter HeLa cells by inducing macropinocytic uptake. Using confocal microscopy, live‐cell imaging, targeted RNAi screening and perturbants of endosome maturation, we analyzed the properties and maturation pathway of the macropinocytic vacuoles containing VACV MVs in HeLa cells. The vacuoles first acquired markers of early endosomes [Rab5, early endosome antigen 1 and phosphatidylinositol(3)P]. Prior to release of virus cores into the cytoplasm, they contained markers of late endosomes and lysosomes (Rab7a, lysosome‐associated membrane protein 1 and sorting nexin 3). RNAi screening of endocytic cell factors emphasized the importance of late compartments for VACV infection. Follow‐up perturbation analysis showed that infection required Rab7a and PIKfyve, confirming that VACV is a late‐penetrating virus dependent on macropinosome maturation. VACV EV infection was inhibited by depletion of many of the same factors, indicating that both infectious particle forms share the need for late vacuolar conditions for penetration. 相似文献
4.
Yuna Kurokawa Akane Yoshida Emi Fujii Kanna Tomioku Hiroki Hayashi Kenji Tanabe Akikazu Fujita 《Traffic (Copenhagen, Denmark)》2019,20(1):82-95
Phosphatidylinositol 4‐phophate (PtdIns(4)P) is an essential signaling molecule in the Golgi body, endosomal system, and plasma membrane and functions in the regulation of membrane trafficking, cytoskeletal organization, lipid metabolism and signal transduction pathways, all mediated by direct interaction with PtdIns(4)P‐binding proteins. PtdIns(4)P was recently reported to have functional roles in autophagosome biogenesis. LC3 and GABARAP subfamilies and a small GTP‐binding protein, Rab7, are localized on autophagosomal membranes and participate at each stage of autophagosome formation and maturation. To better understand autophagosome biogenesis, it is essential to determine the localization of PtdIns(4)P and to examine its relationship with LC3 and GABARAP subfamilies and Rab7. To analyze PtdIns(4)P distribution, we used an electron microscopy technique that labels PtdIns(4)P on the freeze‐fracture replica of intracellular biological membranes, which minimizes the possibility of artificial perturbation because molecules in the membrane are physically immobilized in situ. Using this technique, we found that PtdIns(4)P is localized on the cytoplasmic, but not the luminal (exoplasmic), leaflet of the inner and outer membranes of autophagosomes. Double labeling revealed that PtdIns(4)P mostly colocalizes with Rab7, but not with LC3B, GABARAP, GABARAPL1 and GABARAPL2. Rab7 plays essential roles in autophagosome maturation and in autophagosome‐lysosome fusion events. We suggest that PtdIns(4)P is localized to the cytoplasmic leaflet of the autophagosome at later stages, which may illuminate the importance of PtdIns(4)P at the later stages of autophagosome formation. 相似文献
5.
Starvation‐induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome–lysosome fusion 下载免费PDF全文
Autophagy, the process for recycling cytoplasm in the lysosome, depends on membrane trafficking. We previously identified Drosophila Sbf as a Rab21 guanine nucleotide exchange factor (GEF) that acts with Rab21 in endosomal trafficking. Here, we show that Sbf/MTMR13 and Rab21 have conserved functions required for starvation‐induced autophagy. Depletion of Sbf/MTMR13 or Rab21 blocked endolysosomal trafficking of VAMP8, a SNARE required for autophagosome–lysosome fusion. We show that starvation induces Sbf/MTMR13 GEF and RAB21 activity, as well as their induced binding to VAMP8 (or closest Drosophila homolog, Vamp7). MTMR13 is required for RAB21 activation, VAMP8 interaction and VAMP8 endolysosomal trafficking, defining a novel GEF‐Rab‐effector pathway. These results identify starvation‐responsive endosomal regulators and trafficking that tunes membrane demands with changing autophagy status. 相似文献
6.
N‐Cadherin Regulates Cell Migration Through a Rab5‐Dependent Temporal Control of Macropinocytosis 下载免费PDF全文
Meng‐Hsuan Wen Jen‐Yeu Wang Yu‐Ting Chiu Mei‐Pin Wang Sue‐Ping Lee Chin‐Yin Tai 《Traffic (Copenhagen, Denmark)》2016,17(7):769-785
Macropinocytosis is a clathrin‐independent endocytic pathway implicated in fluid uptake, pathogen invasion and cell migration. During collective cell migration, macropinocytosis occurs primarily at membrane ruffles arising from the leading edges of migrating cells. We report here that N‐cadherin (Ncad) regulates the tempo of macropinocytosis and thereby influences wound‐induced collective cell migration. Using live‐cell and super‐resolution imaging techniques, we observed that Ncad formed clusters at the membrane ruffles and macropinosomes. De‐clustering of Ncad by an interfering antibody impaired the recruitment of Rab5‐an early endosomal marker‐to the macropinosomes. Moreover, we demonstrated that Ncad interacts with Rab5, and laser ablation of Ncad caused Rab5 to dissociate from the macropinosomes. Although Rab5 detached from macropinosomes upon the de‐clustering of Ncad, the recruitment of late endosomal marker Rab7 occurred earlier. Consequently, both centripetal trafficking of macropinosomes and collective migration were accelerated due to de‐clustering of Ncad. Thus, our results suggest that Ncad is involved in the maturation of macropinocytosis through Rab5 recruitment, linking macropinocytosis and cell migration through a novel function of Ncad. 相似文献
7.
Jun Kashiwazaki Tomoko Iwaki Kaoru Takegawa Chikashi Shimoda Taro Nakamura 《Traffic (Copenhagen, Denmark)》2009,10(7):912-924
Small guanine triphosphatases (GTPases) of the Rab family are key regulators of membrane trafficking events between the various subcellular compartments in eukaryotic cells. Rab7 is a conserved protein required in the late endocytic pathway and in lysosome biogenesis. A Schizosaccharomyces pombe ( S. pombe ) homolog of Rab7, Ypt7, is necessary for trafficking from the endosome to the vacuole and for homotypic vacuole fusion. Here, we identified and characterized a second fission yeast Rab7 homolog, Ypt71. Ypt71 is localized to the vacuolar membrane. Cells deleted for ypt71 + exhibit normal growth rates and morphology. Interestingly, a ypt71 null mutant contains large vacuoles in contrast with the small fragmented vacuoles found in the ypt7 null mutant. Furthermore, the ypt71 mutation does not enhance or alleviate the temperature sensitivity or vacuole fusion defect of ypt7 Δ cells. Like ypt7 Δ cells, overexpression of ypt71 + caused fragmentation of vacuoles and inhibits vacuole fusion under hypotonic conditions. Thus, the two S. pombe Rab7 homologs act antagonistically in regulating vacuolar morphology. Analysis of a chimeric Ypt7/Ypt71 protein showed that Rab7-directed vacuole dynamics, fusion versus fission, largely depends on the medial region of the protein, including a part of RabSF3/α3-L7. 相似文献
8.
Ana Kucera Marita Borg Distefano Axel Berg‐Larsen Frode Skjeldal Urska Repnik Oddmund Bakke Cinzia Progida 《Traffic (Copenhagen, Denmark)》2016,17(3):211-229
Rab9 is a small GTPase that localizes to the trans‐Golgi Network (TGN) and late endosomes. Its main function has long been connected to the recycling of mannose‐6‐phosphate receptors (MPRs). However, recent studies link Rab9 also to autophagy and lysosome biogenesis. In this paper, using confocal imaging, we characterize for the first time the live dynamics of the Rab9 constitutively active mutant, Rab9Q66L. We find that it localizes predominantly to late endosomes and that its expression in HeLa cells disperses TGN46 and cation‐independent (CI‐MPR) away from the Golgi yet, has no effect on the retrograde transport of CI‐MPR. We also show that CI‐MPR and Rab9 enter the endosomal pathway together at the transition stage between early, Rab5‐positive, and late, Rab7a‐positive, endosomes. CI‐MPR localizes transiently to separate domains on these endosomes, where vesicles carrying CI‐MPR attach and detach within seconds. Taken together, our results demonstrate that Rab9 mediates the delivery of CI‐MPR to the endosomal pathway, entering the maturing endosome at the early‐to‐late transition. 相似文献
9.
10.
Clathrin‐mediated endocytosis (CME) and clathrin‐independent endocytosis (CIE) co‐exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6‐associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6‐GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6‐GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin‐coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6‐GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation. 相似文献
11.
Ferlins Show Tissue‐Specific Expression and Segregate as Plasma Membrane/Late Endosomal or Trans‐Golgi/Recycling Ferlins 下载免费PDF全文
Gregory M. I. Redpath Reece A. Sophocleous Lynne Turnbull Cynthia B. Whitchurch Sandra T. Cooper 《Traffic (Copenhagen, Denmark)》2016,17(3):245-266
Ferlins are a family of transmembrane‐anchored vesicle fusion proteins uniquely characterized by 5–7 tandem cytoplasmic C2 domains, Ca2+‐regulated phospholipid‐binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb‐girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca2+‐dependent, vesicle‐mediated membrane repair and otoferlin mutations cause non‐syndromic deafness due to defective Ca2+‐triggered auditory neurotransmission. In this study, we describe the tissue‐specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D‐structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7‐positive late endosomes, supporting potential roles in the late‐endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans‐Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans‐Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type‐I and type‐II ferlins segregate as PM/late‐endosomal or trans‐Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations. 相似文献
12.
13.
14.
Helen L. Johns Claudia Gonzalez‐Lopez Charlotte L. Sayers Michael Hollinshead Gillian Elliott 《Traffic (Copenhagen, Denmark)》2014,15(2):157-178
Herpes simplex virus 1 (HSV1) is an enveloped virus that uses undefined transport carriers for trafficking of its glycoproteins to envelopment sites. Screening of an siRNA library against 60 Rab GTPases revealed Rab6 as the principal Rab involved in HSV1 infection, with its depletion preventing Golgi‐to‐plasma membrane transport of HSV1 glycoproteins in a pathway used by several integral membrane proteins but not the luminal secreted protein Gaussia luciferase. Knockdown of Rab6 reduced virus yield to 1% and inhibited capsid envelopment, revealing glycoprotein exocytosis as a prerequisite for morphogenesis. Rab6‐dependent virus production did not require the effectors myosin‐II, bicaudal‐D, dynactin‐1 or rabkinesin‐6, but was facilitated by ERC1, a factor involved in linking microtubules to the cell cortex. Tubulation and exocytosis of Rab6‐positive, glycoprotein‐containing membranes from the Golgi was substantially augmented by infection, resulting in enhanced and targeted delivery to cell tips. This reveals HSV1 morphogenesis as one of the first biological processes shown to be dependent on the exocytic activity of Rab6. 相似文献
15.
Zhang XM Walsh B Mitchell CA Rowe T 《Biochemical and biophysical research communications》2005,335(1):154-161
Ypt/Rabs are Ras-related GTPases that function as key regulators of intracellular vesicular trafficking. Their slow intrinsic rates of GTP hydrolysis are catalyzed by GTPase-activating proteins (GAPs). Ypt/Rab-GAPs constitute a family of proteins that contain a TBC (Tre-2/Bub2/Cdc16) domain. Only three of the 51 family members predicted in the human genome are confirmed Ypt/Rab-GAPs. Here, we report the identification and characterization of a novel mammalian Ypt/Rab-GAP, TBC domain family, member 15 (TBC1D15). TBC1D15 is ubiquitously expressed and localized predominantly to the cytosol. The TBC domain of TBC1D15 exhibits relatively high homology with that of Gyp7p, a yeast Ypt/Rab-GAP. Furthermore, TBC1D15 stimulates the intrinsic GTPase activity of Rab7, and to a lesser extent Rab11, but is essentially inactive towards Rab4 or Rab6. These data increase the number of mammalian TBC domain family members with demonstrated Rab-GAP activity to four, and suggest that TBC1D15 may be involved in Rab7-mediated late endosomal trafficking. 相似文献
16.
Jenny C. Schafer Nicholas W. Baetz Lynne A. Lapierre Rebecca E. McRae Joseph T. Roland James R. Goldenring 《Traffic (Copenhagen, Denmark)》2014,15(3):292-308
A tripartite association of Rab11a with both Rab11‐FIP2 and MYO5B regulates recycling endosome trafficking. We sought to define the intermolecular interactions required between Rab11‐FIP2 and MYO5B. Using a random mutagenesis strategy, we identified point mutations at S229P or G233E in Rab11‐FIP2 that caused loss of interaction with MYO5B in yeast two‐hybrid assays as well as loss of interaction of Rab11‐FIP2(129‐356) with MYO5B tail when expressed in HeLa cells. Single mutations or the double S229P/G233E mutation failed to alter the association of full‐length Rab11‐FIP2 with MYO5B tail in HeLa cells. While EGFP‐Rab11‐FIP2 wild type colocalized with endogenous MYO5B staining in MDCK cells, EGFP‐Rab11‐FIP2(S229P/G233E) showed a significant decrease in localization with endogenous MYO5B. Analysis of Rab11a‐containing vesicle movement in live HeLa cells demonstrated that when the MYO5B/Rab11‐FIP2 association is perturbed by mutation or by Rab11‐FIP2 knockdown, vesicle movement is increased in both speed and track length, consistent with an impairment of MYO5B tethering at the cytoskeleton. These results support a critical role for the interaction of MYO5B with Rab11‐FIP2 in stabilizing the functional complex with Rab11a, which regulates dynamic movements of membrane recycling vesicles. 相似文献
17.
Phillip A. Vanlandingham Brian P. Ceresa 《The Journal of biological chemistry》2009,284(18):12110-12124
The small molecular weight G-protein RAB7 is localized to both early and
late endosomes and has been shown to be critical for trafficking through the
endocytic pathway. The role of RAB7 in the endocytic pathway has been
controversial, with some groups reporting that it regulates trafficking from
early to late endosomes and others ascribing its role to trafficking between
late endosomes and lysosomes. In this study, we use RNA interference to
identify the exact step RAB7 regulates in the movement of the epidermal growth
factor receptor (EGFR) from the cell surface to the lysosome. In the absence
of RAB7, trafficking of the EGF·EGFR complex through the early endosome
to the late endosome/multivesicular body (LE/MVB) does not change, but exiting
from the LE/MVB is blocked. Ultrastructural analysis reveals that RAB7 is not
required for formation of intraluminal vesicles of the LE/MVB, since
RAB7-deficient cells have an increased number of enlarged LE/MVBs densely
packed with intraluminal vesicles. Biochemical data indicate that the EGFR
complex is sequestered in these intraluminal vesicles. Together, these data
provide evidence that RAB7 is required for the transfer of cargo from the
LE/MVB to the lysosome and for endocytic organelle maintenance.The endocytic pathway regulates a number of fundamental cellular processes.
These include the uptake of nutrients, immune response, intracellular
transport, and regulation of cell surface receptor signaling
(1). Disruption of normal
endocytic trafficking can affect cellular homeostasis and lead to changes in
cell physiology that range from hyperproliferation to cell death.
Understanding the molecular regulation of endocytic trafficking will provide a
better understanding of basic cell biology as well as identify potential
molecular targets for diseases characterized by defects in endocytic
trafficking.By following the postinternalization events of cell surface receptors,
considerable work has been done to elucidate the molecular details of the
endocytic pathway (2). Many
cell surface receptors, either constitutively or in response to ligand, use
this degradative pathway to regulate receptor and/or ligand levels. Following
clathrin-mediated internalization, the endocytic pathway is composed of a
series of dynamic stages that progressively shuttle cargo from clathrin-coated
vesicles to early endosomes, to late endosomes/multivesicular bodies
(LE/MVBs),2 and
finally to lysosomes for degradation. Each of these endocytic stages is
defined by the morphology and protein composition of the organelle.Endocytic trafficking is coordinated by a variety of proteins that regulate
endosome maturation, movement, fission, and fusion. Primary among these are
the small molecular weight G-proteins called RABs
(3). Rab proteins are members
of the Ras superfamily of GTPases that cycle between GTP-bound active and
GDP-bound inactive states. The nucleotide bound state of the RAB determines
whether it can interact with downstream effectors. Individual RAB proteins
have been shown to act as hubs that regulate distinct trafficking steps
temporally and spatially by facilitating vesicle motility, tethering, and
fusion (4,
5).Rab7 localizes to both the early endosome and the LE/MVB and has been shown
to be a necessary component of endocytic trafficking and lysosomal degradation
(6). However, there is no
consensus as to the exact molecular function of RAB7 in the endocytic pathway.
Some reports have implicated RAB7 in regulating cargo movement out of early
endosomes
(7–10),
whereas others have reported it to function in the more distal process of
lysosomal delivery from LE/MVBs
(11,
12). Live cell imaging
indicates that RAB7 replaces RAB5 as cargo is trafficked through endocytic
compartments (10,
13). However, it remains
unclear if the presence of RAB7 indicates that it is immediately functional or
if it is positioning itself to be used later in the endocytic pathway.
Alternatively, as has been proposed in Caenorhabditis elegans, Rab7
may regulate multiple endocytic steps
(14).Previous attempts to understand the function of RAB7 have relied primarily
on overexpression of wild type or mutant RAB7
(11,
12,
15,
16). This approach carries the
caveat that high levels of the exogenous protein increase the potential for
nonphysiological interactions between an overexpressed RAB and downstream RAB
effectors. This concern was highlighted by a recent analysis that showed
promiscuity between a variety of RABs and RAB effectors
(17). To overcome these
issues, we have used the alternative approach of depleting endogenous RAB7
with siRNA and examining EGF·EGFR endocytic trafficking in the absence
of RAB7.In this study, we show that RAB7 is required for lysosomal degradation of
the EGF·EGFR complex. Upon dissecting the endocytic pathway of
RAB7-deficient cells, we find that cargo can proceed through EEA1 (early
endosome antigen 1)-positive
early endosomes and into CD63-positive LE/MVB. However, in the absence of
RAB7, the EGF·EGFR complex does not exit the LE/MVB and is retained in
its intraluminal vesicles. This disrupted trafficking is mirrored by an
altered equilibrium between the endocytic organelles, as indicated by the
accumulation of enlarged, densely packed LE/MVB and a decrease in the size and
number of lysosomes. Based on these data, we have generated a model that RAB7
is dispensable for EGFR endocytic trafficking from the cell surface to the
intraluminal vesicles of the LE/MVB but is required for fusion of the LE/MVB
and the lysosome. 相似文献
18.
Elodie Prince Benjamin Kroeger Dragan Gligorov Clive Wilson Suzanne Eaton Franois Karch Marko Brankatschk Robert K. Maeda 《Traffic (Copenhagen, Denmark)》2019,20(2):137-151
The male seminal fluid contains factors that affect female post‐mating behavior and physiology. In Drosophila, most of these factors are secreted by the two epithelial cell types that make up the male accessory gland: the main and secondary cells. Although secondary cells represent only ~4% of the cells of the accessory gland, their contribution to the male seminal fluid is essential for sustaining the female post‐mating response. To better understand the function of the secondary cells, we investigated their molecular organization, particularly with respect to the intracellular membrane transport machinery. We determined that large vacuole‐like structures found in the secondary cells are trafficking hubs labeled by Rab6, 7, 11 and 19. Furthermore, these organelles require Rab6 for their formation and many are essential in the process of creating the long‐term postmating behavior of females. In order to better serve the intracellular membrane and protein trafficking communities, we have created a searchable, online, open‐access imaging resource to display our complete findings regarding Rab localization in the accessory gland. 相似文献
19.
Roya M. Dayam Amra Saric Ryan E. Shilliday Roberto J. Botelho 《Traffic (Copenhagen, Denmark)》2015,16(9):1010-1026
Macrophages internalize and sequester pathogens into a phagosome. Phagosomes then sequentially fuse with endosomes and lysosomes, converting into degradative phagolysosomes. Phagosome maturation is a complex process that requires regulators of the endosomal pathway including the phosphoinositide lipids. Phosphatidylinositol‐3‐phosphate and phosphatidylinositol‐3,5‐bisphosphate (PtdIns(3,5)P2), which respectively control early endosomes and late endolysosomes, are both required for phagosome maturation. Inhibition of PIKfyve, which synthesizes PtdIns(3,5)P2, blocked phagosome–lysosome fusion and abated the degradative capacity of phagosomes. However, it is not known how PIKfyve and PtdIns(3,5)P2 participate in phagosome maturation. TRPML1 is a PtdIns(3,5)P2‐gated lysosomal Ca2+ channel. Because Ca2+ triggers membrane fusion, we postulated that TRPML1 helps mediate phagosome–lysosome fusion. Using Fcγ receptor‐mediated phagocytosis as a model, we describe our research showing that silencing of TRPML1 hindered phagosome acquisition of lysosomal markers and reduced the bactericidal properties of phagosomes. Specifically, phagosomes isolated from TRPML1‐silenced cells were decorated with lysosomes that docked but did not fuse. We could rescue phagosome maturation in TRPML1‐silenced and PIKfyve‐inhibited cells by forcible Ca2+ release with ionomycin. We also provide evidence that cytosolic Ca2+ concentration increases upon phagocytosis in a manner dependent on TRPML1 and PIKfyve. Overall, we propose a model where PIKfyve and PtdIns(3,5)P2 activate TRPML1 to induce phagosome–lysosome fusion. 相似文献
20.
Amulya Priya Inna V Kalaidzidis Yannis Kalaidzidis David Lambright Sunando Datta 《Traffic (Copenhagen, Denmark)》2015,16(1):68-84
Retromer, a peripheral membrane protein complex, plays an instrumental role in host of cellular processes by its ability to recycle receptors from endosomes to the trans‐Golgi network. It consists of two distinct sub‐complexes, a membrane recognizing, sorting nexins (SNX) complex and a cargo recognition, vacuolar protein sorting (Vps) complex. Small GTPase, Rab7 is known to recruit retromer on endosomal membrane via interactions with the Vps sub‐complex. The molecular mechanism underlying the recruitment process including the role of individual Vps proteins is yet to be deciphered. In this study, we developed a FRET‐based assay in HeLa cells that demonstrated the interaction of Rab7 with Vps35 and Vps26 in vivo. Furthermore, we showed that Rab7 recruits retromer to late endosomes via direct interactions with N‐terminal conserved regions in Vps35. However, the single point mutation, which disrupts the interaction between Vps35 and Vps26, perturbed the Rab7‐mediated recruitment of retromer in HeLa cells. Using biophysical measurements, we demonstrate that the association of Vps26 with Vps35 resulted in high affinity binding between the Vps sub‐complex and the activated Rab7 suggesting for a possible allosteric role of Vps26. Thus, this study provides molecular insights into the essential role of Vps26 and Vps35 in Rab7‐mediated recruitment of the core retromer complex. 相似文献