GLUT4 and Transferrin Receptor Are Differentially Sorted Along the Endocytic Pathway in CHO Cells

Wei, Maria L.; Bonzelius, Frank; Scully, Rebecca; Kelly, Regis B.; Herman, Gary

doi:10.1083/jcb.140.3.565

Cited by 54 publications

(72 citation statements)

References 82 publications

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“…Such cells have been used in various studies to investigate the mechanism whereby insulin stimulation promotes GLUT4 translocation to the plasma membrane [23,26,27,30,31]. The CHO cells, like 3T3-L1 adipocytes, possess GLUT4 storage vesicles which translocate to the plasma membrane in response to insulin stimulation.…”

Section: Discussionmentioning

confidence: 99%

“…Such cells are known to show insulin-stimulated glucose transport and to share many similarities with 3T3-L1 adipocytes, a canonical cell line used to investigate insulin-stimulated glucose transport [26,27]. The cells were cultured in DMEM as this has been shown to be required for insulin-responsive trafficking [27].…”

Section: Endogenous Munc18c Associates With Endogenous Pkcζmentioning

confidence: 99%

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Protein kinase-ζ interacts with munc18c: role in GLUT4 trafficking

2005

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Aims/hypothesis: Insulin-stimulated glucose transport requires a signalling cascade through kinases protein kinase (PK) Cζ/λ and PKB that leads to movement of GLUT4 vesicles to the plasma membrane. The aim of this study was to identify missing links between the upstream insulin-regulated kinases and the GLUT4 vesicle trafficking system. Materials and methods: A yeast twohybrid screen was conducted, using as bait full-length mouse munc18c, a protein known to be part of the GLUT4 vesicle trafficking machinery. Results: The yeast twohybrid screen identified PKCζ as a novel interactor with munc18c. Glutathione S transferase (GST) pull-downs with GST-tagged munc18c constructs confirmed the interaction, mapped a key region of munc18c that binds PKCζ to residues 295-338 and showed that the N-terminal region of PKCζ was required for the interaction. Endogenous munc18c was shown to associate with endogenous PKCζ in vivo in various cell types. Importantly, insulin stimulation increased the association by approximately three-fold. Moreover, disruption of PKCζ binding to munc18c by deletion of residues 295-338 of munc18c or deletion of the N-terminal region of PKCζ markedly inhibited the ability of insulin to stimulate glucose uptake or GLUT4 translocation. Conclusions/interpretation: We have identified a physiological interaction between munc18c and PKCζ that is insulin-regulated. This establishes a link between a kinase (PKCζ) involved in the insulin signalling cascade and a known component of the GLUT4 vesicle trafficking pathway (munc18c). The results indicate that PKCζ regulates munc18c and suggest a model whereby insulin triggers the docking of PKCζ to munc18c, resulting in enhanced GLUT4 translocation to the plasma membrane.

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Section: Discussionmentioning

confidence: 99%

Section: Endogenous Munc18c Associates With Endogenous Pkcζmentioning

confidence: 99%

Protein kinase-ζ interacts with munc18c: role in GLUT4 trafficking

2005

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“…After washing, cells were fixed for 20 min at 4°C in 4% paraformaldehyde and processed for immunofluorescence as described previously (20,21).…”

Section: Methodsmentioning

confidence: 99%

AP-3-dependent Mechanisms Control the Targeting of a Chloride Channel (ClC-3) in Neuronal and Non-neuronal Cells

Salazar¹,

Love²,

Styers³

et al. 2004

Journal of Biological Chemistry

108

129

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Adaptor protein (AP)-2 and AP-3-dependent mechanisms control the sorting of membrane proteins into synaptic vesicles. Mouse models deficient in AP-3, mocha, develop a neurological phenotype of which the central feature is an alteration of the luminal synaptic vesicle composition. This is caused by a severe reduction of vesicular levels of the zinc transporter 3 (ZnT3). It is presently unknown whether this mocha defect is restricted to ZnT3 or encompasses other synaptic vesicle proteins capable of modifying synaptic vesicle contents, such as transporters or channels. In this study, we identified a chloride channel, ClC-3, whose level in synaptic vesicles and hippocampal mossy fiber terminals was reduced in the context of the mocha AP-3 deficiency. In PC-12 cells, ClC-3 was present in transferrin receptorpositive endosomes, where it was targeted to synapticlike microvesicles (SLMV) by a mechanism sensitive to brefeldin A, a signature of the AP-3-dependent route of SLMV biogenesis. ClC-3 was packed in SLMV along with the AP-3-targeted synaptic vesicle protein ZnT3. Co-segregation of ClC-3 and ZnT3 to common intracellular compartments was functionally significant as revealed by increased vesicular zinc transport with increased ClC3 expression. Our work has identified a synaptic vesicle protein in which trafficking to synaptic vesicles is regulated by AP-3. In addition, our findings indicate that ClC-3 and ZnT3 reside in a common vesicle population where they functionally interact to determine vesicle luminal composition.

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“…After washing, cells were fixed for 20 min at 4°C in 4% paraformaldehyde and processed for immunofluorescence as described previously (Faundez et al, 1997;Wei et al, 1998). Secondary antibodies used were Alexa-conjugated goat anti-mouse 488 and/or goat anti-rabbit 568.…”

Section: Immunolocalizationsmentioning

confidence: 99%

The Zinc Transporter ZnT3 Interacts with AP-3 and It Is Preferentially Targeted to a Distinct Synaptic Vesicle Subpopulation

Salazar

Love

Werner

et al. 2004

MBoC

109

170

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Synaptic vesicles (SV) are generated by two different mechanisms, one AP-2 dependent and one AP-3 dependent. It has been uncertain, however, whether these mechanisms generate SV that differ in molecular composition. We explored this hypothesis by analyzing the targeting of ZnT3 and synaptophysin both to PC12 synaptic-like microvesicles (SLMV) as well as SV isolated from wild-type and AP-3-deficient mocha brains. ZnT3 cytosolic tail interacted selectively with AP-3 in cell-free assays. Accordingly, pharmacological disruption of either AP-2-or AP-3-dependent SLMV biogenesis preferentially reduced synaptophysin or ZnT3 targeting, respectively; suggesting that these antigens were concentrated in different vesicles. As predicted, immuno-isolated SLMV revealed that ZnT3 and synaptophysin were enriched in different vesicle populations. Likewise, morphological and biochemical analyses in hippocampal neurons indicated that these two antigens were also present in distinct but overlapping domains. ZnT3 SV content was reduced in AP-3-deficient neurons, but synaptophysin was not altered in the AP-3 null background. Our evidence indicates that neuroendocrine cells assemble molecularly heterogeneous SV and suggests that this diversity could contribute to the functional variety of synapses. INTRODUCTIONThe molecular diversity in total brain synaptic vesicle (SV) composition is generally presumed to result from differential expression of synaptic vesicle membrane proteins in different brain regions. However, the possibility that synaptic vesicles differ in composition because of different biogenesis mechanisms has not been explored. Different vesiculation pathways could result in molecularly diverse synaptic vesicles. Vesiculation mechanisms are known to produce distinct cargo carriers from a population of donor membranes (Bonifacino and Dell'Angelica, 1999;Springer et al., 1999;Boehm and Bonifacino, 2001). This process is achieved by adaptor complexes that recognize and concentrate specific membrane proteins in the donor membranes (Bonifacino and Dell 'Angelica, 1999;Kirchhausen, 1999). PC12 cells have been shown to possess two such adaptor-dependent pathways for the assembly of synaptic vesicles, also known as synaptic-like microvesicles (SLMV) (Shi et al., 1998;de Wit et al., 1999;Jarousse and Kelly, 2001). In one pathway, SLMV are generated from the plasma membrane by a vesiculation mechanism that requires the adaptor AP-2, clathrin, and the GTPase dynamin (Shi et al., 1998). In the second pathway, SLMV are generated from endosomes by the adaptor complex AP-3 (Faundez et al., 1998;Blumstein et al., 2001) and the GTPase ARF1 (Faundez et al., 1997). In contrast to the plasma membrane pathway, the endosomederived mechanism is highly sensitive to brefeldin A (BFA) (Shi et al., 1998).Phenotypes observed in the AP-3-deficient mocha mouse are consistent with a role for the AP-3-dependent, endosome-derived pathway in neurons (Kantheti et al., 1998). The mocha mossy fibers are devoid of both the synaptic vesiclespecific zinc transpor...

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GLUT4 and Transferrin Receptor Are Differentially Sorted Along the Endocytic Pathway in CHO Cells

Cited by 54 publications

References 82 publications

Protein kinase-ζ interacts with munc18c: role in GLUT4 trafficking

Protein kinase-ζ interacts with munc18c: role in GLUT4 trafficking

AP-3-dependent Mechanisms Control the Targeting of a Chloride Channel (ClC-3) in Neuronal and Non-neuronal Cells

The Zinc Transporter ZnT3 Interacts with AP-3 and It Is Preferentially Targeted to a Distinct Synaptic Vesicle Subpopulation

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