scholarly journals Discovery of a vezatin-like protein for dynein-mediated early endosome transport

2015 ◽  
Vol 26 (21) ◽  
pp. 3816-3827 ◽  
Author(s):  
Xuanli Yao ◽  
Herbert N. Arst ◽  
Xiangfeng Wang ◽  
Xin Xiang
Keyword(s):  
Aspergillus Nidulans ◽  
Genetic Study ◽  
Cytoplasmic Dynein ◽  
Early Endosome ◽  
Dependent Manner ◽  
C Terminus ◽  
Early Endosomes ◽  
Abnormal Accumulation ◽  
Hyphal Tip

Early endosomes are transported bidirectionally by cytoplasmic dynein and kinesin-3, but how the movements are regulated in vivo remains unclear. Here our forward genetic study led to the discovery of VezA, a vezatin-like protein in Aspergillus nidulans, as a factor critical for early endosome distribution. Loss of vezA causes an abnormal accumulation of early endosomes at the hyphal tip, where microtubule plus ends are located. This abnormal accumulation depends on kinesin-3 and is due to a decrease in the frequency but not the speed of dynein-mediated early endosome movement. VezA-GFP signals are enriched at the hypha tip in an actin-dependent manner but are not obviously associated with early endosomes, thus differing from the early endosome association of the cargo adapter HookA (Hook in A. nidulans). On loss of VezA, HookA associates normally with early endosomes, but the interaction between dynein-dynactin and the early-endosome-bound HookA is significantly decreased. However, VezA is not required for linking dynein-dynactin to the cytosolic ∆C-HookA, lacking the cargo-binding C-terminus. These results identify VezA as a novel regulator required for the interaction between dynein and the Hook-bound early endosomes in vivo.

scholarly journals The p25 subunit of the dynactin complex is required for dynein–early endosome interaction

2011 ◽  
Vol 193 (7) ◽  
pp. 1245-1255 ◽  
Author(s):  
Jun Zhang ◽  
Xuanli Yao ◽  
Lauren Fischer ◽  
Juan F. Abenza ◽  
Miguel A. Peñalva ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Cytoplasmic Dynein ◽  
Early Endosome ◽  
Physical Interaction ◽  
Nuclear Distribution ◽  
Early Endosomes ◽  
Pointed End ◽  
Dynactin Complex ◽  
Hyphal Tip

Cytoplasmic dynein transports various cellular cargoes including early endosomes, but how dynein is linked to early endosomes is unclear. We find that the Aspergillus nidulans orthologue of the p25 subunit of dynactin is critical for dynein-mediated early endosome movement but not for dynein-mediated nuclear distribution. In the absence of NUDF/LIS1, p25 deletion abolished the localization of dynein–dynactin to the hyphal tip where early endosomes abnormally accumulate but did not prevent dynein–dynactin localization to microtubule plus ends. Within the dynactin complex, p25 locates at the pointed end of the Arp1 filament with Arp11 and p62, and our data suggest that Arp11 but not p62 is important for p25–dynactin association. Loss of either Arp1 or p25 significantly weakened the physical interaction between dynein and early endosomes, although loss of p25 did not apparently affect the integrity of the Arp1 filament. These results indicate that p25, in conjunction with the rest of the dynactin complex, is important for dynein–early endosome interaction.

scholarly journals The BLOC-1 complex promotes endosomal maturation by recruiting the Rab5 GTPase-activating protein Msb3

2013 ◽  
Vol 201 (1) ◽  
pp. 97-111 ◽  
Author(s):  
Arun T. John Peter ◽  
Jens Lachmann ◽  
Meenakshi Rana ◽  
Madeleine Bunge ◽  
Margarita Cabrera ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Sorting ◽  
Early Endosome ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Gtpase Activating Protein ◽  
Early Endosomes ◽  
Lysosome Related Organelles

Membrane microcompartments of the early endosomes serve as a sorting and signaling platform, where receptors are either recycled back to the plasma membrane or forwarded to the lysosome for destruction. In metazoan cells, three complexes, termed BLOC-1 to -3, mediate protein sorting from the early endosome to lysosomes and lysosome-related organelles. We now demonstrate that BLOC-1 is an endosomal Rab-GAP (GTPase-activating protein) adapter complex in yeast. The yeast BLOC-1 consisted of six subunits, which localized interdependently to the endosomes in a Rab5/Vps21-dependent manner. In the absence of BLOC-1 subunits, the balance between recycling and degradation of selected cargoes was impaired. Additionally, our data show that BLOC-1 is both a Vps21 effector and an adapter for its GAP Msb3. BLOC-1 and Msb3 interacted in vivo, and both mutants resulted in a redistribution of active Vps21 to the vacuole surface. We thus conclude that BLOC-1 controls the lifetime of active Rab5/Vps21 and thus endosomal maturation along the endocytic pathway.

scholarly journals HookA is a novel dynein–early endosome linker critical for cargo movement in vivo

2014 ◽  
Vol 204 (6) ◽  
pp. 1009-1026 ◽  
Author(s):  
Jun Zhang ◽  
Rongde Qiu ◽  
Herbert N. Arst ◽  
Miguel A. Peñalva ◽  
Xin Xiang
Keyword(s):  
Aspergillus Nidulans ◽  
Filamentous Fungus ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Early Endosome ◽  
Genetic Screen ◽  
Binding Domain ◽  
Terminal Part ◽  
Microtubule Binding Domain

Cytoplasmic dynein transports membranous cargoes along microtubules, but the mechanism of dynein–cargo interaction is unclear. From a genetic screen, we identified a homologue of human Hook proteins, HookA, as a factor required for dynein-mediated early endosome movement in the filamentous fungus Aspergillus nidulans. HookA contains a putative N-terminal microtubule-binding domain followed by coiled-coil domains and a C-terminal cargo-binding domain, an organization reminiscent of cytoplasmic linker proteins. HookA–early endosome interaction occurs independently of dynein–early endosome interaction and requires the C-terminal domain. Importantly, HookA interacts with dynein and dynactin independently of HookA–early endosome interaction but dependent on the N-terminal part of HookA. Both dynein and the p25 subunit of dynactin are required for the interaction between HookA and dynein–dynactin, and loss of HookA significantly weakens dynein–early endosome interaction, causing a virtually complete absence of early endosome movement. Thus, HookA is a novel linker important for dynein–early endosome interaction in vivo.

scholarly journals SMAP2, a Novel ARF GTPase-activating Protein, Interacts with Clathrin and Clathrin Assembly Protein and Functions on the AP-1–positive Early Endosome/Trans-Golgi Network

2006 ◽  
Vol 17 (6) ◽  
pp. 2592-2603 ◽  
Author(s):  
Waka Natsume ◽  
Kenji Tanabe ◽  
Shunsuke Kon ◽  
Naomi Yoshida ◽  
Toshio Watanabe ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Adaptor Proteins ◽  
Early Endosome ◽  
Dependent Manner ◽  
Transferrin Receptors ◽  
Gtpase Activating Protein ◽  
Trans Golgi Network ◽  
Early Endosomes ◽  
Golgi Network

We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1–dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking.

scholarly journals FHIP and FTS proteins are critical for dynein-mediated transport of early endosomes in Aspergillus

2014 ◽  
Vol 25 (14) ◽  
pp. 2181-2189 ◽  
Author(s):  
Xuanli Yao ◽  
Xiangfeng Wang ◽  
Xin Xiang
Keyword(s):  
Aspergillus Nidulans ◽  
Protein Level ◽  
Cytoplasmic Dynein ◽  
Early Endosome ◽  
Human Homologue ◽  
Genome Wide ◽  
A Genome ◽  
Early Endosomes ◽  
Microtubule Motor

The minus end–directed microtubule motor cytoplasmic dynein transports various cellular cargoes, including early endosomes, but how dynein binds to its cargo remains unclear. Recently fungal Hook homologues were found to link dynein to early endosomes for their transport. Here we identified FhipA in Aspergillus nidulans as a key player for HookA (A. nidulans Hook) function via a genome-wide screen for mutants defective in early-endosome distribution. The human homologue of FhipA, FHIP, is a protein in the previously discovered FTS/Hook/FHIP (FHF) complex, which contains, besides FHIP and Hook proteins, Fused Toes (FTS). Although this complex was not previously shown to be involved in dynein-mediated transport, we show here that loss of either FhipA or FtsA (A. nidulans FTS homologue) disrupts HookA–early endosome association and inhibits early endosome movement. Both FhipA and FtsA associate with early endosomes, and interestingly, while FtsA–early endosome association requires FhipA and HookA, FhipA–early endosome association is independent of HookA and FtsA. Thus FhipA is more directly linked to early endosomes than HookA and FtsA. However, in the absence of HookA or FtsA, FhipA protein level is significantly reduced. Our results indicate that all three proteins in the FtsA/HookA/FhipA complex are important for dynein-mediated early endosome movement.

scholarly journals CLIP-170 Homologue and NUDE Play Overlapping Roles in NUDF Localization in Aspergillus nidulans

2006 ◽  
Vol 17 (4) ◽  
pp. 2021-2034 ◽  
Author(s):  
Vladimir P. Efimov ◽  
Jun Zhang ◽  
Xin Xiang
Keyword(s):  
Aspergillus Nidulans ◽  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Live Cells ◽  
Physiological Conditions ◽  
Physical Interactions ◽  
Cytoplasmic Microtubules ◽  
Growth Promoting ◽  
Green Fluorescent ◽  
Hyphal Tip

Proteins in the cytoplasmic dynein pathway accumulate at the microtubule plus end, giving the appearance of comets when observed in live cells. The targeting mechanism for NUDF (LIS1/Pac1) of Aspergillus nidulans, a key component of the dynein pathway, has not been clear. Previous studies have demonstrated physical interactions of NUDF/LIS1/Pac1 with both NUDE/NUDEL/Ndl1 and CLIP-170/Bik1. Here, we have identified the A. nidulans CLIP-170 homologue, CLIPA. The clipA deletion did not cause an obvious nuclear distribution phenotype but affected cytoplasmic microtubules in an unexpected manner. Although more microtubules failed to undergo long-range growth toward the hyphal tip at 32°C, those that reached the hyphal tip were less likely to undergo catastrophe. Thus, in addition to acting as a growth-promoting factor, CLIPA also promotes microtubule dynamics. In the absence of CLIPA, green fluorescent protein-labeled cytoplasmic dynein heavy chain, p150Glued dynactin, and NUDF were all seen as plus-end comets at 32°C. However, under the same conditions, deletion of both clipA and nudE almost completely abolished NUDF comets, although nudE deletion itself did not cause a dramatic change in NUDF localization. Based on these results, we suggest that CLIPA and NUDE both recruit NUDF to the microtubule plus end. The plus-end localization of CLIPA itself seems to be regulated by different mechanisms under different physiological conditions. Although the KipA kinesin (Kip2/Tea2 homologue) did not affect plus-end localization of CLIPA at 32°C, it was required for enhancing plus-end accumulation of CLIPA at an elevated temperature (42°C).

scholarly journals Endocytic Down-Regulation of ErbB2 Is Stimulated by Cleavage of Its C-Terminus

2007 ◽  
Vol 18 (9) ◽  
pp. 3656-3666 ◽  
Author(s):  
Mads Lerdrup ◽  
Silas Bruun ◽  
Michael V. Grandal ◽  
Kirstine Roepstorff ◽  
Malene M. Kristensen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Retention Mechanism ◽  
Full Length ◽  
Dependent Manner ◽  
Lysosomal Degradation ◽  
Down Regulation ◽  
C Terminus ◽  
Early Endosomes

High ErbB2 levels are associated with cancer, and impaired endocytosis of ErbB2 could contribute to its overexpression. Therefore, knowledge about the mechanisms underlying endocytic down-regulation of ErbB2 is warranted. The C-terminus of ErbB2 can be cleaved after various stimuli, and after inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the N- and C-terminus of ErbB2, respectively (YFP-ErbB2-CFP). After geldanamycin stimulation YFP-ErbB2-CFP became cleaved in nonapoptotic cells in a proteasome-dependent manner, and a markedly larger relative amount of cleaved YFP-ErbB2-CFP was observed in early endosomes than in the plasma membrane. Furthermore, cleavage took place at the plasma membrane, and cleaved ErbB2 was internalized and degraded far more efficiently than full-length ErbB2. Concordantly, a C-terminally truncated ErbB2 was also readily endocytosed and degraded in lysosomes compared with full-length ErbB2. Altogether, we suggest that geldanamycin leads to C-terminal cleavage of ErbB2, which releases the receptor from a retention mechanism and causes endocytosis and lysosomal degradation of ErbB2.

scholarly journals Di-Leucine Signals Mediate Targeting of Tyrosinase and Synaptotagmin to Synaptic-like Microvesicles within PC12 Cells

1999 ◽  
Vol 10 (11) ◽  
pp. 3979-3990 ◽  
Author(s):  
Anastasiya D. Blagoveshchenskaya ◽  
Eric W. Hewitt ◽  
Daniel F. Cutler
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Protein Traffic ◽  
C Terminus ◽  
Targeting Signal

One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRP-tyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents.

scholarly journals LIS1 regulates cargo-adapter–mediated activation of dynein by overcoming its autoinhibition in vivo

2019 ◽  
Vol 218 (11) ◽  
pp. 3630-3646 ◽  
Author(s):  
Rongde Qiu ◽  
Jun Zhang ◽  
Xin Xiang
Keyword(s):  
Aspergillus Nidulans ◽  
Mechanism Of Action ◽  
Developmental Disorder ◽  
Model Organism ◽  
Cytoplasmic Dynein ◽  
Binding Domain ◽  
Significant Extent ◽  
Experimental Systems ◽  
Microtubule Motor

Deficiency of the LIS1 protein causes lissencephaly, a brain developmental disorder. Although LIS1 binds the microtubule motor cytoplasmic dynein and has been linked to dynein function in many experimental systems, its mechanism of action remains unclear. Here, we revealed its function in cargo-adapter–mediated dynein activation in the model organism Aspergillus nidulans. Specifically, we found that overexpressed cargo adapter HookA (Hook in A. nidulans) missing its cargo-binding domain (ΔC-HookA) causes dynein and its regulator dynactin to relocate from the microtubule plus ends to the minus ends, and this relocation requires LIS1 and its binding protein, NudE. Astonishingly, the requirement for LIS1 or NudE can be bypassed to a significant extent by mutations that prohibit dynein from forming an autoinhibited conformation in which the motor domains of the dynein dimer are held close together. Our results suggest a novel mechanism of LIS1 action that promotes the switch of dynein from the autoinhibited state to an open state to facilitate dynein activation.

scholarly journals Phosphorylation of α-dystrobrevin is essential for αkap accumulation and acetylcholine receptor stability

2020 ◽  
Vol 295 (31) ◽  
pp. 10677-10688
Author(s):  
Po-Ju Chen ◽  
Diego Zelada ◽  
Dina Cheryne Belhasan ◽  
Mohammed Akaaboune
Keyword(s):  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Tyrosine Residues ◽  
C Terminus ◽  
The Stability ◽  
High Level

The maintenance of a high density of the acetylcholine receptor (AChR) is the hallmark of the neuromuscular junction. Muscle-specific anchoring protein (αkap) encoded within the calcium/calmodulin-dependent protein kinase IIα (CAMK2A) gene is essential for the maintenance of AChR clusters both in vivo and in cultured muscle cells. The underlying mechanism by which αkap is maintained and regulated remains unknown. Here, using human cell lines, fluorescence microscopy, and pulldown and immunoblotting assays, we show that α-dystrobrevin (α-dbn), an intracellular component of the dystrophin glycoprotein complex, directly and robustly promotes the stability of αkap in a concentration-dependent manner. Mechanistically, we found that the phosphorylatable tyrosine residues of α-dbn are essential for the stability of α-dbn itself and its interaction with αkap, with substitution of three tyrosine residues in the α-dbn C terminus with phenylalanine compromising the αkap–α-dbn interaction and significantly reducing both αkap and α-dbn accumulation. Moreover, the αkap–α-dbn interaction was critical for αkap accumulation and stability. We also found that the absence of either αkap or α-dbn markedly reduces AChRα accumulation and that overexpression of α-dbn or αkap in cultured muscle cells promotes the formation of large agrin-induced AChR clusters. Collectively, these results indicate that the stability of αkap and α-dbn complex plays an important role in the maintenance of high-level expression of AChRs.

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