Localization of SMAP2 to the TGN and its Function in the Regulation of TGN Protein Transport

The formation of clathrin-coated vesicles is essential for intracellular membrane trafficking between subcellular compartments and is triggered by the ARF family of small GTPases. We previously identified SMAP1 as an ARF6 GTPase-activating protein that functions in clathrin-dependent endocytosis. Because abnormalities in clathrin-dependent trafficking are often associated with oncogenesis, we targeted Smap1 in mice to examine its physiological and pathological significance. Smap1-deficent mice exhibited healthy growth, but their erythroblasts showed enhanced transferrin endocytosis. In mast cells cultured in SCF, Smap1 deficiency did not affect the internalization of c-KIT but impaired the sorting of internalized c-KIT from multivesicular bodies to lysosomes, resulting in intracellular accumulation of undegraded c-KIT that was accompanied by enhanced activation of ERK and increased cell growth. Interestingly, approximately 50% of aged Smap1-deficient mice developed anemia associated with morphologically dysplastic cells of erythroid-myeloid lineage, which are hematological abnormalities similar to myelodysplastic syndrome (MDS) in humans. Furthermore, some Smap1-deficient mice developed acute myeloid leukemia (AML) of various subtypes. Collectively, to our knowledge these results provide the first evidence in a mouse model that the deregulation of clathrin-dependent membrane trafficking may be involved in the development of MDS and subsequent AML. IntroductionIntracellular and extracellular homeostasis is maintained by a vesicle transport system that mediates the trafficking of membrane proteins to appropriate organelles. Clathrin-coated vesicles are formed at donor membrane sites in a highly ordered manner, and a number of molecules are involved in this process. Among them, small GTPases of the ARF family play a central role in vesicle formation. An ARF molecule cycles between two conformations, an active GTP-bound form and an inactive GDP-bound form. This cycling is mediated by a guanine nucleotide exchange factor that replaces GDP with GTP and a GTPase-activating protein (GAP) that hydrolyzes GTP to GDP and converts ARF into its inactive form. There are 6 ARFs (ARF1-ARF6) and several ARF-related proteins in mammals (1, 2). ARF6 is an isoform that localizes mainly to the plasma membrane and functions in the endocytosis and recycling of vesicles as well as in actin rearrangement and lipid metabolism (3,4).We previously demonstrated that small ARF GAP1 (referred to as SMAP1) is a regulator of clathrin-dependent endocytosis, based on a series of observations (5, 6). First, SMAP1 exhibits GAP activity against ARF6, as assessed by an in vitro GAP assay. Second, SMAP1 localizes to juxta-plasma membrane regions in which ARF6 also exists. Third, SMAP1 binds to the clathrin heavy chain directly via its clathrin-binding box. Fourth, overexpression of SMAP1 abrogates clathrin-dependent internalization of the transferrin receptor and E-cadherin.

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“…MEFs were prepared according to a previously published procedure (49). Briefly, 16.5-day-old embryos were isolated from pregnant mice.…”

Section: Methodsmentioning

confidence: 99%

Smap1 deficiency perturbs receptor trafficking and predisposes mice to myelodysplasia

et al. 2013

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“…Given that SMAP2 partly localized to the TGN in addition to its primary localization in the recycling endosomes, SMAP2 might inactivate Arf1 on the TGN membranes. In fact, SMAP2-knockout mouse embryonic fibroblasts (MEFs) show increased levels of Arf1-GTP (Funaki et al, 2011). Interestingly, depletion of CHC, AP-1γ or evectin-2, which all lead to mislocalization of SMAP2 from recycling endosomes, also increased the Arf1-GTP levels (supplementary material Fig.…”

Section: Discussionmentioning

confidence: 99%

Transport of cholera toxin B-subunit from recycling endosomes to the Golgi requires clathrin and AP-1

Matsudaira

Niki

Taguchi

et al. 2015

Journal of Cell Science

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The retrograde pathway is defined by the transport of proteins and lipids from the plasma membrane through endosomes to the Golgi complex, and is essential for a variety of cellular activities. Recycling endosomes are important sorting stations for some retrograde cargo. SMAP2, a GTPase-activating protein (GAP) for Arf1 with a putative clathrin-binding domain, has previously been shown to participate in the retrograde transport of the cholera toxin B-subunit (CTxB) from recycling endosomes. Here, we found that clathrin, a vesicle coat protein, and clathrin adaptor protein complex 1 (AP-1) were present at recycling endosomes and were needed for the retrograde transport of CTxB from recycling endosomes to the Golgi, but not from the plasma membrane to recycling endosomes. SMAP2 immunoprecipitated clathrin and AP-1 through a putative clathrin-binding domain and a CALM-binding domain, and SMAP2 mutants that did not interact with clathrin or AP-1 could not localize to recycling endosomes. Moreover, knockdown of Arf1 suppressed the retrograde transport of CTxB from recycling endosomes to the Golgi. These findings suggest that retrograde transport is mediated by clathrin-coated vesicles from recycling endosomes and that the role of the coat proteins is in the recruitment of Arf GAP to transport vesicles.

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“…The cells were maintained in 5% CO 2 in air at 37°C. The procedure for preparing mouse embryo fibroblasts (MEF) was described previously [13,14]. Briefly, male and female mice with the Smap1(f/f);Smap2(+/À) genotype were mated, and El5.5 embryos were collected from pregnant mice.…”

Section: Cell Culturementioning

confidence: 99%

“…For example, SMAP1 and SMAP2 prefer Arf6 and Arf1 as substrates, respectively [10,11]. Thus, SMAP1 is involved in the Arf6-dependent endocytosis of the transferrin receptor and E-cadherin [10,12], whereas SMAP2 is involved in the Arf1-dependent membrane trafficking between early endosomes and the trans-Golgi network (TGN) [11,13]. In addition, a gene targeting study revealed that Smap1 deficiency predisposes mice to myelodysplastic syndrome due to impaired sorting of endocytosed c-Kit from multivesicular bodies to lysosomes [14].…”

Section: Introductionmentioning

confidence: 99%

The Arf GTPase-activating protein SMAP1 promotes transferrin receptor endocytosis and interacts with SMAP2

Kobayashi

Kon

Henmi

et al. 2014

Biochemical and Biophysical Research Communications

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Localization of SMAP2 to the TGN and its Function in the Regulation of TGN Protein Transport

Cited by 11 publications

References 37 publications

Smap1 deficiency perturbs receptor trafficking and predisposes mice to myelodysplasia

Smap1 deficiency perturbs receptor trafficking and predisposes mice to myelodysplasia

Transport of cholera toxin B-subunit from recycling endosomes to the Golgi requires clathrin and AP-1

The Arf GTPase-activating protein SMAP1 promotes transferrin receptor endocytosis and interacts with SMAP2

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