Raft association of SNAP receptors acting in apical trafficking in Madin-Darby canine kidney cells

Abstract

We have investigated the relationships between the apical sorting mechanism using lipid rafts and the soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) machinery, which is involved in membrane docking and fusion. We first confirmed that anti-alpha-SNAP antibodies inhibit the apical pathway in Madin- Darby canine kidney (MDCK) cells; in addition, we report that a recombinant SNAP protein stimulates the apical transport whereas a SNAP mutant inhibits this transport step. Based on t-SNARE overexpression experiments and the effect of botulinum neurotoxin E, syntaxin 3 and SNAP-23 have been implicated in apical membrane trafficking. Here, we show in permeabilized MDCK cells that antisyntaxin 3 and anti-SNAP-23 antibodies lower surface delivery of an apical reporter protein. Moreover, using a similar approach, we show that tetanus toxin-insensitive, vesicle-associated membrane protein (TI-VAMP; also called VAMP7), a recently described apical v-SNARE, is involved. Furthermore, we show the presence of syntaxin 3 and TI-VAMP in isolated apical carriers. Polarized apical sorting has been postulated to be mediated by the clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We provide evidence that syntaxin 3 and TI-VAMP are raft-associated. These data support a raft-based mechanism for the sorting of not only apically destined cargo but also of SNAREs having functions in apical membrane-docking and fusion events.

Figure 1

Alpha-SNAP involvement in apical membrane trafficking. (A) Dose-dependent decrease of the TGN to the apical and to the basolateral plasma membrane transport by antibodies against alpha-SNAP. The transport was monitored in SLO-permeabilized, filter-grown MDCK cells that were infected with either influenza virus or VSV. HA and VSV G were used as apical and basolateral markers, respectively. Note that the basolateral transport is more affected than the apical transport by the addition of the 3E2 antibody. (B) Influence of the His₆-tagged wild-type and dominant inhibitory mutant (L294A) recombinant alpha-SNAP proteins on apical delivery.

Figure 2

SNAP-23, syntaxin 3, and TI-VAMP involvement in the apical pathway. (A) Effect on the apical transport of HA of antibodies against SNAP-23 and syntaxin 11, 4, and 3 at the respective concentrations of 1.6, 1.5, 1.5, and 1.3 μM. (B) Distribution of TI-VAMP in polarized MDCK cells. Confocal (x,z) section of fixed cells stained for TI-VAMP (green) and DNA (propidium iodide; red). Cells were observed on a Zeiss LSM 510 confocal microscope. (Bar = 15 μm.) (C) Dose-dependent reduction of TGN to apical surface transport by the anti-TI-VAMP antibody.

Figure 3

Syntaxin 3 and TI-VAMP localization in isolated apical carriers. Immunoelectron micrographs of isolated apical carriers. (A) HA (10 nm gold) and syntaxin 3 (5 nm gold). (B) HA (10 nm gold) and TI-VAMP (5 nm gold). (Bar = 50 nm.)

Figure 4

Raft association of syntaxin 3 and TI-VAMP. Filter-grown MDCK cells were infected with influenza virus and chased at 20°C before scraping. (A) Postnuclear supernatant (PNS) extracted with Triton X-100 on ice were floated in a two-step gradient. (B) PNS either treated with MβCD (+MβCD) or not were extracted with Triton X-100 on ice, and membranes were floated in an OptiPrep multistep gradient. (C) Apical TGN-derived carriers either treated with MβCD (+MβCD) or not were extracted with Triton X-100 on ice, and membranes were floated in an OptiPrep multistep gradient. Fractions were collected from the top, and precipitated proteins were analyzed by Western blotting. The first three fractions correspond to the volume of the sample submitted to floatation.