Mapmodulin, cytoplasmic dynein, and microtubules enhance the transport of mannose 6-phosphate receptors from endosomes to the trans-golgi network

Abstract

Late endosomes and the Golgi complex maintain their cellular localizations by virtue of interactions with the microtubule-based cytoskeleton. We study the transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network in vitro. We show here that this process is facilitated by microtubules and the microtubule-based motor cytoplasmic dynein; transport is inhibited by excess recombinant dynamitin or purified microtubule-associated proteins. Mapmodulin, a protein that interacts with the microtubule-associated proteins MAP2, MAP4, and tau, stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact Chinese hamster ovary cells. The present study shows that mapmodulin also stimulates the initial rate with which mannose 6-phosphate receptors are transported from late endosomes to the trans-Golgi network in vitro. These findings represent the first indication that mapmodulin can stimulate a vesicle transport process, and they support a model in which the microtubule-based cytoskeleton enhances the efficiency of vesicle transport between membrane-bound compartments in mammalian cells.

Figure 1

Nocodazole inhibits the initial rate of MPR transport from endosomes to the TGN in vitro. Reactions were carried out for the indicated times at 37°C in the presence (filled circles) or absence (open circles) of nocodazole (10 μg/ml) and 0.2 mg/ml cytosolic proteins and 100 ng/ml α-SNAP. Values shown represent the average of triplicate determinations; bars represent SD.

Figure 2

Mapmodulin stimulates the initial rate of MPR transport from endosomes to the TGN in vitro. (A) Reactions contained 0.2 mg/ml cytosol, 100 ng/ml α-SNAP, 50 ng/ml Rab9–GDI complex, and either no mapmodulin (−) or 50 ng/ml (0.6 nM) mapmodulin (+) and were incubated at 30°C for the indicated times. (B) Kinetics of endosome-to-TGN transport in low or full cytosol. Reactions contained 0.2 mg/ml cytosol, 100 ng/ml α-SNAP (low cytosol), or 1 mg/ml cytosol (full cytosol) and were incubated for 5 or 10 min at 30°C. (C) Purified mapmodulin was titrated in the presence of low cytosol as described in B; reactions were for 10 min. Cytosol-dependent transport was determined by subtracting transport measured in the absence of cytosol. Values shown represent the average of duplicate determinations; bars represent SD.

Figure 3

Proteins used in this study. Coomassie blue–stained SDS-PAGE (7.5%) of (A) cytoplasmic dynein purified from bovine brain (A) (DHC, dynein heavy chain; DIC, dynein intermediate chain; DLICs, dynein light intermediate chains), recombinant dynamitin (B) (**), and heat-stable MAPs purified from bovine brain (C) (MAP2 and tau).

Figure 4

Cytoplasmic dynein stimulates the initial rate of MPR transport from endosomes to the TGN. Reactions were carried out as described in Figure 1B with increasing concentrations of cytoplasmic dynein. Cytosol-dependent transport was obtained by subtracting transport measured in the absence of cytosol. Shown are the averages of duplicate determinations; bars represent SD.

Figure 5

Recombinant dynamitin inhibits MPR transport from endosomes to the TGN. Purified recombinant dynamitin was added to reactions containing 1 mg/ml cytosol. Transport is given relative to control reactions containing equal amounts of BSA. Cytosol-dependent transport was obtained by subtracting transport measured in the absence of cytosol. Shown are average and SDs of two independent experiments carried out in duplicate.

Figure 6

Heat-stable MAPs inhibit MPR transport from endosomes to the TGN. Purified heat-stable MAPs were added to reactions containing 1 mg/ml cytosol. Transport is given relative to control reactions containing equal amounts of BSA. Cytosol-dependent transport was obtained by subtracting transport measured in the absence of cytosol. Shown are averages of duplicates; bars represent SD.