A novel Rab9 effector required for endosome-to-TGN transport

Abstract

Rab9 GTPase is required for the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network in living cells, and in an in vitro system that reconstitutes this process. We have used the yeast two-hybrid system to identify proteins that interact preferentially with the active form of Rab9. We report here the discovery of a 40-kD protein (p40) that binds Rab9-GTP with roughly fourfold preference to Rab9-GDP. p40 does not interact with Rab7 or K-Ras; it also fails to bind Rab9 when it is bound to GDI. The protein is found in cytosol, yet a significant fraction (approximately 30%) is associated with cellular membranes. Upon sucrose density gradient flotation, membrane- associated p40 cofractionates with endosomes containing mannose 6-phosphate receptors and the Rab9 GTPase. p40 is a very potent transport factor in that the pure, recombinant protein can stimulate, significantly, an in vitro transport assay that measures transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network. The functional importance of p40 is confirmed by the finding that anti-p40 antibodies inhibit in vitro transport. Finally, p40 shows synergy with Rab9 in terms of its ability to stimulate mannose 6-phosphate receptor transport. These data are consistent with a model in which p40 and Rab9 act together to drive the process of transport vesicle docking.

Figure 1

Discovery of a yeast two hybrid cDNA clone encoding a peptide that preferentially binds Rab9– GTP. β-galactosidase activity of yeast strains co-expressing the clone 361-GAL4 activation domain hybrid and GAL4 DNA binding domain hybrids of either Rab9 (black), Rab9S21N (white), or Rab7 (gray).

Figure 2

cDNA and predicted amino acid sequence of p40. The 1,298-bp cDNA and predicted 372 amino acid protein are shown. The two in frame stop codons are underlined. The portion of the cDNA identified in the two hybrid screen began at base pair 564.

Figure 3

(A) Alignment of kelch repeats in p40. h, Hydrophobic residues; t, turns. Bold residues indicate conservation with the original kelch motif. Also highlighted in bold in p40 are proline residues, whose positions are conserved among p40 repeats but are not present in other kelch-motif proteins. (B) Schematic diagram of the postulated β barrel structure of p40 showing the region of Ral2p similarity (bold) and a large loop domain. The dashed line does not represent additional amino acid sequences. The portion of the protein shown to interact with Rab9 by two hybrid analysis is indicated at the bottom.

Figure 4

Subcellular distribution of p40. (A) Coomassie blue-stained SDS-PAGE of purified, recombinant, His-tagged p40 and immunoblot of cell extracts. Lane 1, recombinant p40 (1 μg); lane 2, K562 cytosol (100 μg); lanes 3 and 4, HeLa cytosol or membranes (each ∼100 μg, or 10% of a 10-cm dish). (B) A portion of p40 cofractionates with Rab9 and mannose 6-phosphate receptors upon sucrose gradient flotation of K562 cell postnuclear supernatant. The top of the gradient is at the left.

Figure 5

Purified p40 binds Rab9–GTP in strong preference to Rab9–GDP. (A) An example of immunoblot binding data obtained is shown. Values shown in B were determined by PhosphorImager quantitation; Rab9 standards were included on the gels to permit determination of the nanogram amounts of Rab9 bound.

Figure 6

p40 inhibits Rab9's GTPase activity but not its rate of nucleotide exchange. (A) Nucleotide exchange was assayed with 10 nM Rab9 and 100 nM p40 according to Soldati et al. (1994). (B) Effect of p40 on the Rab9 GTPase. Reactions contained 50 nM Rab9. Control reactions displayed a rate of 0.0122 min⁻¹, in agreement with previously reported values (Shapiro et al., 1993).

Figure 7

p40 functions in endosome-to-TGN transport. (A) Recombinant p40 was added to reactions containing 0.6 mg/ml cytosol. No additional stimulation was observed at higher p40 concentrations. (B) p40 and Rab9/GDI act synergistically in MPR transport. Recombinant p40 (10 ng/ml), Rab9 in an equimolar complex with GDI (100 ng/ml), or both components were added to transport reactions containing 0.65 mg/ml cytosol. Transport is presented in relation to that seen with 1 mg/ml cytosol (2,345 cpm). Error bars represent standard error of the mean (n = 2).

Figure 8

Anti-p40 antibodies inhibit MPR transport. Immune or preimmune IgG (75 μg/ml) was added to MPR transport reactions containing 1 mg/ml cytosol and where indicated, p40 (100 ng/ml). Error bars represent standard error of the mean (n = 2). Control reactions yielded 724 cpm.

Figure 9

Depletion of cytosolic p40 does not inhibit MPR transport. (A) Anti-p40 immunoblot analysis of cytosols (100 μg each) preadsorbed with either preimmune IgG (control) or anti-p40 immune IgG (depleted). (B) Transport activity of the cytosols shown in A. Cytosols were assayed at 0.4 mg/ml; 100% transport of control cytosol represents 556 cpm. Values shown represent the average of duplicate determinations (standard error ⩽ 5%) in a representative experiment.