Endosomal Protein Trafficking Research Articles

Retromer dysfunction in amyotrophic lateral sclerosis

Retromer is a heteropentameric complex that plays a specialized role in endosomal protein sorting and trafficking. Here, we report a reduction in the retromer proteins-vacuolar protein sorting 35 (VPS35), VPS26A, and VPS29-in patients with amyotrophic lateral sclerosis (ALS) and in the ALS model provided by transgenic (Tg) mice expressing the mutant superoxide dismutase-1 G93A. These changes are accompanied by a reduction of levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluA1, a proxy of retromer function, in spinal cords from Tg SOD1G93A mice. Correction of the retromer deficit by a viral vector expressing VPS35 exacerbates the paralytic phenotype in Tg SOD1G93A mice. Conversely, lowering Vps35 levels in Tg SOD1G93A mice ameliorates the disease phenotype. In light of these findings, we propose that mild alterations in retromer inversely modulate neurodegeneration propensity in ALS.

WASHC1 interacts with MCM2-7 complex to promote cell survival under replication stress.

WASHC1 is a member of the Wiskott-Aldrich syndrome protein (WASP) family and is involved in endosomal protein sorting and trafficking through the generation of filamentous actin (F-actin) via activation of the Arp2/3 complex. There is increasing evidence that WASHC1 is present in the nucleus and nuclear WASHC1 plays important roles in regulating gene transcription, DNA repair as well as maintaining nuclear organization. However, the multi-faceted functions of nuclear WASHC1 still need to be clarified. We show here that WASHC1 interacts with several components of the minichromosome maintenance (MCM) 2-7 complex by using co-immunoprecipitation and in situ proximity ligation assay. WASHC1-depleted cells display normal DNA replication and S-phase progression. However, loss of WASHC1 sensitizes HeLa cells to DNA replication inhibitor hydroxyurea (HU) and increases chromosome instability of HeLa and 3T3 cells under condition of HU-induced replication stress. Re-expression of nuclear WASHC1 in WASHC1KO 3T3 cells rescues the deficiency of WASHC1KO cells in the chromosomal stability after HU treatment. Moreover, chromatin immunoprecipitation assay indicates that WASHC1 associates with DNA replication origins, and knockdown of WASHC1 inhibits MCM protein loading at origins. Since efficient loading of excess MCM2-7 complexes is required for cells to survive replicative stress, these results demonstrate that WASHC1 promotes cell survival and maintain chromosomal stability under replication stress through recruitment of excess MCM complex to origins.

The Seminiferous Epithelial Cycle of Spermatogenesis: Role of Non-receptor Tyrosine Kinases.

Non-receptor tyrosine kinases (NRTKs) are implicated in various biological processes including cell proliferation, differentiation, survival, and apoptosis, as well as cell adhesion and movement. NRTKs are expressed in all mammals and in different cell types, with extraordinarily high expression in the testis. Their association with the plasma membrane and dynamic subcellular localization are crucial parameters in their activation and function. Many NRTKs are found in endosomal protein trafficking pathways, which suggests a novel mechanism to regulate the timely junction restructuring in the mammalian testis to facilitate spermiation and germ cell transport across the seminiferous epithelium.

Receptor-mediated endocytosis 8 (RME-8)/DNAJC13 is a novel positive modulator of autophagy and stabilizes cellular protein homeostasis

The cellular protein homeostasis (proteostasis) network responds effectively to insults. In a functional screen in C. elegans, we recently identified the gene receptor-mediated endocytosis 8 (rme-8; human ortholog: DNAJC13) as a component of the proteostasis network. Accumulation of aggregation-prone proteins, such as amyloid-β 42 (Aβ), α-synuclein, or mutant Cu/Zn-superoxide dismutase (SOD1), were aggravated upon the knockdown of rme-8/DNAJC13 in C. elegans and in human cell lines, respectively. DNAJC13 is involved in endosomal protein trafficking and associated with the retromer and the WASH complex. As both complexes have been linked to autophagy, we investigated the role of DNAJC13 in this degradative pathway. In knockdown and overexpression experiments, DNAJC13 acts as a positive modulator of autophagy. In contrast, the overexpression of the Parkinson’s disease-associated mutant DNAJC13(N855S) did not enhance autophagy. Reduced DNAJC13 levels affected ATG9A localization at and its transport from the recycling endosome. As a consequence, ATG9A co-localization at LC3B-positive puncta under steady-state and autophagy-induced conditions is impaired. These data demonstrate a novel function of RME-8/DNAJC13 in cellular homeostasis by modulating ATG9A trafficking and autophagy.

Identification of Host Trafficking Genes Required for HIV-1 Virological Synapse Formation in Dendritic Cells.

Dendritic cells (DCs) are one of the earliest targets of HIV-1 infection acting as a "Trojan horse," concealing the virus from the innate immune system and delivering it to T cells via virological synapses (VS). To explicate how the virus is trafficked through the cell to the VS and evades degradation, a high-throughput small interfering RNA screen targeting membrane trafficking proteins was performed in monocyte-derived DCs. We identified several proteins including BIN-1 and RAB7L1 that share common roles in transport from endosomal compartments. Depletion of target proteins resulted in an accumulation of virus in intracellular compartments and significantly reduced viral trans-infection via the VS. By targeting endocytic trafficking and retromer recycling to the plasma membrane, we were able to reduce the virus's ability to accumulate at budding microdomains and the VS. Thus, we identify key genes involved in a pathway within DCs that is exploited by HIV-1 to traffic to the VS.IMPORTANCE The lentivirus human immunodeficiency virus (HIV) targets and destroys CD4+ T cells, leaving the host vulnerable to life-threatening opportunistic infections associated with AIDS. Dendritic cells (DCs) form a virological synapse (VS) with CD4+ T cells, enabling the efficient transfer of virus between the two cells. We have identified cellular factors that are critical in the induction of the VS. We show that ADP-ribosylation factor 1 (ARF1), bridging integrator 1 (BIN1), and Rab GTPases RAB7L1 and RAB8A are important regulators of HIV-1 trafficking to the VS and therefore the infection of CD4+ T cells. We found these cellular factors were essential for endosomal protein trafficking and formation of the VS and that depletion of target proteins prevented virus trafficking to the plasma membrane by retaining virus in intracellular vesicles. Identification of key regulators in HIV-1 trans-infection between DC and CD4+ T cells has the potential for the development of targeted therapy to reduce trans-infection of HIV-1 in vivo.

Oncogenic Pathways and Loss of the Rab11 GTPase Synergize To Alter Metabolism in Drosophila.

Colorectal cancer is a complex disease driven by well-established mutations such as APC and other yet to be identified pathways. The GTPase Rab11 regulates endosomal protein trafficking, and previously we showed that loss of Rab11 caused intestinal inflammation and hyperplasia in mice and flies. To test the idea that loss of Rab11 may promote cancer progression, we have analyzed archival human patient tissues and observed that 51 out of 70 colon cancer tissues had lower Rab11 protein staining. By using the Drosophila midgut model, we have found that loss of Rab11 can lead to three changes that may relate to cancer progression. First is the disruption of enterocyte polarity based on staining of the FERM domain protein Coracle. Second is an increased proliferation due to an increased expression of the JAK-STAT pathway ligand Upd3. Third is an increased expression of ImpL2, which is an IGFBP7 homolog and can suppress metabolism. Furthermore, loss of Rab11 can act synergistically with the oncoprotein RasV12 to regulate these cancer-related phenotypes.

Altered dopamine release and monoamine transporters in Vps35 p.D620N knock-in mice

Vacuolar protein sorting 35 (VPS35) is a core component of the retromer trimer required for endosomal membrane-associated protein trafficking. The discovery of a missense mutation, Vps35 p.D620N implicates retromer dysfunction in the pathogenesis of Parkinson’s disease (PD). We have characterized a knock-in mouse with a Vps35 p.D620N substitution (hereafter referred to as VKI) at 3 months of age. Standardized behavioral testing did not observe overt movement disorder. Tyrosine hydroxylase (TH)-positive nigral neuron counts and terminal expression in striata were comparable across genotypes. Fast scan cyclic voltammetry revealed increased dopamine release in VKI striatal slices. While extracellular dopamine collected via striatal microdialysis of freely moving animals was comparable across genotypes, the ratio of dopamine metabolites to dopamine suggests increased dopamine turnover in VKI homozygous mice. Western blot of striatal proteins revealed a genotype-dependent decrease in dopamine transporter (DAT) along with an increase in vesicular monoamine transporter 2 (VMAT2), albeit independent of changes in other synaptic markers. The reduction in DAT was further supported by immunohistochemical analysis. The data show that the dopaminergic system of VKI mice is profoundly altered relative to wild-type littermates. We conclude early synaptic dysfunction contributes to age-related pathophysiology in the nigrostriatal system that may lead to parkinsonism in man.

Endosomal sorting and trafficking, the retromer complex and neurodegeneration.

The retromer is a highly conserved multimeric protein complex present in all eukaryotic cells whose activity is essential for regulating the recycling and retrieval of numerous protein cargos from the endosome to trans-Golgi network or the cell surface. In recent years, molecular and genomic studies have provided evidence that aberrant regulation of endosomal protein sorting and trafficking secondary to a dysfunction of the retromer complex could be implicated in the pathogenesis of several neurodegenerative diseases. Thus, deficiency or mutations in one or more protein components of the retromer leads to increased accumulation of protein aggregates as well as enhanced cellular neurotoxicity. In this review, we will discuss the structure and function of the retromer complex and its neurobiology, its relevance to key molecules involved in neurodegeneration and the potential role that it plays in the development of two major neurodegenerative disorders, Parkinson’s disease and Alzheimer’s disease. Lastly, we will discuss the viability of targeting the retromer via pharmacological chaperones or genetic approaches to enhance or restore its function as a novel and unifying disease-modifying strategy against these diseases.

Extracellular anti-angiogenic proteins augment an endosomal protein trafficking pathway to reach mitochondria and execute apoptosis in HUVECs

Classic endocytosis destinations include the recycling endosome returning to the plasma membrane or the late endosome (LE) merging with lysosomes for cargo degradation. However, the anti-angiogenic proteins angiostatin and isthmin, are endocytosed and trafficked to mitochondria (Mito) to execute apoptosis of endothelial cells. How these extracellular proteins reach mitochondria remains a mystery. Through confocal and super-resolution fluorescent microscopy, we demonstrate that angiostatin and isthmin are trafficked to mitochondria through the interaction between LE and Mito. Using purified organelles, the LE–Mito interaction is confirmed through in vitro lipid-fusion assay, as well as single vesicle total internal reflection fluorescent microscopy. LE–Mito interaction enables the transfer of not only lipids but also proteins from LE to Mito. Angiostatin and isthmin augment this endosomal protein trafficking pathway and make use of it to reach mitochondria to execute apoptosis. Cell fractionation and biochemical analysis identified that the cytosolic scaffold protein Na+/H+ exchanger regulatory factor 1 (NHERF1) associated with LE and the t-SNARE protein synaptosome-associated protein 25 kDa (SNAP25) associated with Mito form an interaction complex to facilitate LE–Mito interaction. Proximity ligation assay coupled with fluorescent microscopy showed that both NHERF1 and SNAP25 are located at the contacting face between LE and Mito. RNAi knockdown of either NHERF1 or SNAP25 suppressed not only the mitochondrial trafficking of angiostatin and isthmin but also their anti-angiogenic and pro-apoptotic functions. Hence, this study reveals a previously unrealized endosomal protein trafficking pathway from LE to Mito that allows extracellular proteins to reach mitochondria and execute apoptosis.

Sorting Out the Role of α-Synuclein in Retromer-Mediated Endosomal Protein Sorting.

Retromer is a phylogenetically conserved, multisubunit coat complex that controls endosomal protein trafficking and sorting. Mutations in the retromer gene VPS35 cause late-onset Parkinson disease, suggesting that trafficking defects cause neurodegeneration. Sorting nexins assist retromer to guide cell surface proteins to their assigned destinations, and our interest here is sorting nexin 3 (Snx3). Snx3 binds to membranes via a phox homolog (PX) domain that binds phosphatidylinositol 3-phosphate (PI3P), and in human cells its cargo proteins are the transferrin and Wnt receptors and the divalent metal ion transporter, whereas in yeast the best characterized cargo is the iron permease Ftr1. We recently discovered that α-synuclein inhibits Snx3-retromer recycling of Ftr1 in an unexpected way: α-synuclein, which avidly binds to negatively charged lipids, blocks the association of Snx3 to early endosomes. Here, we discuss mechanisms by which α-synuclein can disrupt Snx3-retromer–mediated recycling.

Effect of fish oil on agonist-induced receptor internalization of the PG F2α receptor and cell signaling in bovine luteal cells in vitro

Many receptors span the plasma membrane allowing for signal transduction, converting extracellular signals into intracellular signals. Following ligand-induced activation, membrane-bound receptors are taken into endocytic vesicles, where they are targeted for degradation or recycled back to the plasma membrane. The objectives of the present study were to determine the influence of fish oil on (1) PGF2α-induced receptor internalization and trafficking of the PGF2α (FP) receptor, (2) cytoskeletal structural integrity, and (3) PGF2α-induced mitogen-activated protein kinase (MAPK) signaling in bovine luteal cells. Bovine ovaries were obtained from a local abattoir and corpora lutea (CL; n = 4–6) were digested using collagenase. For all experiments, cells were incubated in either BSA or fish oil–supplemented media for 72 h to allow incorporation of omega-3 fatty acids into biological membranes. Confocal microscopy was used to determine the influence of fish oil on PGF2α-induced receptor internalization and trafficking of the FP receptor and cytoskeletal structural integrity. In addition, Western blotting was used to determine the effects of fish oil on PGF2α-induced MAPK signaling in bovine luteal cells. Results from the present study demonstrate that fish oil disrupts the colocalization of Gαq with both caveola microdomains and FP receptor as well as PGF2α-induced MAPK signaling. This disruption of the FP receptor with the G-protein alpha subunit may be one mechanism by which a MAPK signaling is diminished following the addition of PGF2α. Furthermore, fish oil disrupts FP receptor internalization and endosomal protein trafficking without detectable changes in the cytoskeleton.

Activation of TPA-response element present in human Lemur Tyrosine Kinase 2 (lmtk2) gene increases its expression

Regulatory elements present in the promoter of a gene drive the expression of the gene in response to various stimuli. Lemur Tyrosine Kinase 2 (LMTK2) is a membrane-anchored Serine/Threonine kinase involved in endosomal protein trafficking and androgen signaling amongst other processes. Previous studies have shown this protein to be of therapeutic importance in cystic fibrosis and prostate cancer. However, nothing is known about the endogenous expression of this protein and its regulation. In this study, we analyzed the gene encoding human LMTK2, to look for possible regulatory elements that could affect its expression. Interestingly, the human lmtk2 gene contains a consensus TPA (12- O-Tetradecanoylphorbol-13-acetate)-responsive element (TRE) in the region preceding its start codon. The element with the sequence TGAGTCA modulates LMTK2 expression in response to treatment with TPA, a synthetic Protein Kinase C (PKC) activator. It serves as the binding site for c-Fos, a member of the Activator Protein −1 (AP-1) transcription factor complex, which is transactivated by PKC. We observed that TPA, at low concentrations, increases the promoter activity of LMTK2, which leads to a subsequent increase in the mRNA transcript and protein levels. This modulation occurs through binding of the AP-1 transcription factor complex to the lmtk2 promoter. Thus, our current study has established LMTK2 as a TPA-responsive element-containing gene, which is upregulated downstream of PKC activation. Considering the involvement of LMTK2 in intracellular processes as well as pathological conditions, our findings demonstrate a way to modulate intracellular LMTK2 levels pharmacologically for potentially therapeutic purposes.

Retromer

Lucas and Hierro introduce the retromer and its role in endosomal protein sorting and trafficking.

TMEM106B, a frontotemporal lobar dementia (FTLD) modifier, associates with FTD-3-linked CHMP2B, a complex of ESCRT-III.

BackgroundTransmembrane protein 106B (TMEM106B) has been identified as a risk factor for frontotemporal lobar degeneration, which is the second most common form of progressive dementia in people under 65 years of age. Mutations in charged multivesicular body protein 2B (CHMP2B), which is involved in endosomal protein trafficking, have been found in chromosome 3-linked frontotemporal dementia. Despite the number of studies on both CHMP2B and TMEM106B in the endolysosomal pathway, little is known about the relationship between CHMP2B and TMEM106B in the endosomal/autophagy pathway.ResultsThis study found that endogenous TMEM106B was partially sequestered in CHMP2B-positive structures, suggesting its possible involvement in endosomal sorting complexes required for transport (ESCRT)-associated pathways. The role of single nucleotide polymorphisms of TMEM106B (T185, S185, or S134N) in the ESCRT-associated pathways were characterized. The T185 and S185 variants were more localized to Rab5-/Rab7-positive endosomes compared with S134N, while all of the variants were more localized to Rab7-positive endosomes compared to Rab5-positive endosomes. T185 was more associated with CHMP2B compared to S185. Autophagic flux was slightly reduced in the T185-expressing cells compared to the control or S185-expressing cells. Moreover, T185 slightly enhanced the accumulation of EGFR, impairments in autophagic flux, and neurotoxicity that were caused by CHMP2BIntron5 compared to S185-expressing cells.ConclusionsThese findings suggest that the T185 variant functions as a risk factor in neurodegeneration with endolysosomal defects. This study provides a better understanding of pathogenic functions of TMEM106B, which is a risk factor for the progression of neurodegenerative diseases that are associated with endosomal defects in the aged brain.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0177-z) contains supplementary material, which is available to authorized users.

Mechanisms Of Fructose Absorption

Mechanisms Of Fructose Absorption

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 modulates endosomal pH and protein trafficking in astrocytes: Relevance to MLC disease pathogenesis

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in the gene encoding MLC1, a membrane protein mainly expressed in astrocytes in the central nervous system. Although MLC1 function is unknown, evidence is emerging that it may regulate ion fluxes. Using biochemical and proteomic approaches to identify MLC1 interactors and elucidate MLC1 function we found that MLC1 interacts with the vacuolar ATPase (V-ATPase), the proton pump that regulates endosomal acidity. Because we previously showed that in intracellular organelles MLC1 directly binds Na, K-ATPase, which controls endosomal pH, we studied MLC1 endosomal localization and trafficking and MLC1 effects on endosomal acidity and function using human astrocytoma cells overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. We found that WT MLC1 is abundantly expressed in early (EEA1+, Rab5+) and recycling (Rab11+) endosomes and uses the latter compartment to traffic to the plasma membrane during hyposmotic stress. We also showed that WT MLC1 limits early endosomal acidification and influences protein trafficking in astrocytoma cells by stimulating protein recycling, as revealed by FITC-dextran measurement of endosomal pH and transferrin protein recycling assay, respectively. WT MLC1 also favors recycling to the plasma-membrane of the TRPV4 cation channel which cooperates with MLC1 to activate calcium influx in astrocytes during hyposmotic stress. Although MLC disease-causing mutations differentially affect MLC1 localization and trafficking, all the mutated proteins fail to influence endosomal pH and protein recycling. This study demonstrates that MLC1 modulates endosomal pH and protein trafficking suggesting that alteration of these processes contributes to MLC pathogenesis.

A Congenital Neutrophil Defect Syndrome Associated with Mutations in VPS45

BackgroundNeutrophils are the predominant phagocytes that provide protection against bacterial and fungal infections. Genetically determined neutrophil disorders confer a predisposition to severe infections and reveal novel mechanisms that control vesicular trafficking, hematopoiesis, and innate immunity.MethodsWe clinically evaluated seven children from five families who had neutropenia, neutrophil dysfunction, bone marrow fibrosis, and nephromegaly. To identify the causative gene, we performed homozygosity mapping using single-nucleotide polymorphism arrays, whole-exome sequencing, immunoblotting, immunofluorescence, electron microscopy, a real-time quantitative polymerase–chain-reaction assay, immunohistochemistry, flow cytometry, fibroblast motility assays, measurements of apoptosis, and zebrafish models. Correction experiments were performed by transfecting mutant fibroblasts with the nonmutated gene.ResultsAll seven affected children had homozygous mutations (Thr224Asn or Glu238Lys, depending on the child's ethnic origin) in VPS45, which encodes a protein that regulates membrane trafficking through the endosomal system. The level of VPS45 protein was reduced, as were the VPS45 binding partners rabenosyn-5 and syntaxin-16. The level of β1 integrin was reduced on the surface of VPS45-deficient neutrophils and fibroblasts. VPS45-deficient fibroblasts were characterized by impaired motility and increased apoptosis. A zebrafish model of vps45 deficiency showed a marked paucity of myeloperoxidase-positive cells (i.e., neutrophils). Transfection of patient cells with nonmutated VPS45 corrected the migration defect and decreased apoptosis.ConclusionsDefective endosomal intracellular protein trafficking due to biallelic mutations in VPS45 underlies a new immunodeficiency syndrome involving impaired neutrophil function. (Funded by the National Human Genome Research Institute and others.)

De-Regulation of JNK and JAK/STAT Signaling in ESCRT-II Mutant Tissues Cooperatively Contributes to Neoplastic Tumorigenesis

Multiple genes involved in endocytosis and endosomal protein trafficking in Drosophila have been shown to function as neoplastic tumor suppressor genes (nTSGs), including Endosomal Sorting Complex Required for Transport-II (ESCRT-II) components vacuolar protein sorting 22 (vps22), vps25, and vps36. However, most studies of endocytic nTSGs have been done in mosaic tissues containing both mutant and non-mutant populations of cells, and interactions among mutant and non-mutant cells greatly influence the final phenotype. Thus, the true autonomous phenotype of tissues mutant for endocytic nTSGs remains unclear. Here, we show that tissues predominantly mutant for ESCRT-II components display characteristics of neoplastic transformation and then undergo apoptosis. These neoplastic tissues show upregulation of c-Jun N-terminal Kinase (JNK), Notch, and Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signaling. Significantly, while inhibition of JNK signaling in mutant tissues partially inhibits proliferation, inhibition of JAK/STAT signaling rescues other aspects of the neoplastic phenotype. This is the first rigorous study of tissues predominantly mutant for endocytic nTSGs and provides clear evidence for cooperation among de-regulated signaling pathways leading to tumorigenesis.

Regulation of WASH-Dependent Actin Polymerization and Protein Trafficking by Ubiquitination

Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a role for ubiquitination in this process. We find that the E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 in retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex.

Membrane-associated RING-CH 10 (MARCH10 Protein) Is a Microtubule-associated E3 Ubiquitin Ligase of the Spermatid Flagella

Spermiogenesis is a complex and dynamic process of the metamorphosis of spermatids into spermatozoa. There is a great deal that is still unknown regarding the regulatory mechanisms for the formation of the sperm flagellum. In this study, we determined that the membrane-associated RING-CH 10 (March10) gene is predominantly expressed in rat testis. We isolated two March10 isoforms encoding MARCH10a and MARCH10b, which are generated by alternative splicing. MARCH10a is a long RING finger protein, and MARCH10b is a short RING finger-less protein. Immunohistochemical staining revealed that the MARCH10 proteins are specifically expressed in elongating and elongated spermatids, and the expression is absent in epididymal spermatozoa. MARCH10 immunoreactivity was observed in the cytoplasmic lobes as well as the principal piece and annulus of the flagella. When overexpressed in COS7 cells, MARCH10a was localized along the microtubules, whereas MARCH10b was distributed throughout the cytoplasm. An in vitro microtubule cosedimentation assay showed that MARCH10a is directly associated with microtubules. An in vitro ubiquitination assay demonstrated that the RING finger domain of MARCH10a exhibits an E3 ubiquitin ligase activity along with the E2 ubiquitin-conjugating enzyme UBE2B. Moreover, MARCH10a undergoes proteasomal degradation by autoubiquitination in transfected COS7 cells, but this activity was abolished upon microtubule disassembly. These results suggest that MARCH10 is involved in spermiogenesis by regulating the formation and maintenance of the flagella in developing spermatids.