Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes

Abstract

Rab11 is a small GTP-binding protein that in cultured mammalian cells has been shown to be concentrated in the pericentriolar endosomal recycling compartment and to play a key role in passage of the recycling transferrin receptor through that compartment [Ullrich, O., Reinsch, S., Urbé, S., Zerial, M. & Parton, R. G. (1996) J. Cell Biol. 135, 913-924]. To obtain insights into the site(s) of action of rab11 within the recycling pathway, we have now compared the effects on recycling at 37 degreesC of overexpression of wild-type rab11 and various mutant forms of this protein in cells that had been loaded with transferrin at either 37 degreesC or 16 degreesC. We show that incubation at 16 degreesC blocks passage of endocytosed transferrin into the recycling compartment and that, whereas the rab11 dominant negative mutant form (S25N) inhibits transferrin recycling after interiorization at either temperature, the wild-type rab11 and constitutively active mutant (Q70L) have no inhibitory effect on the recycling of molecules that were interiorized at 16 degreesC. This differential inhibitory effect shows that two distinct pathways for recycling are followed by the bulk of the transferrin molecules interiorized at the two different temperatures. The incapacity of the constitutively active form of rab11 (Q70L) to inhibit recycling of molecules interiorized at 16 degreesC is consistent with their recycling taking place directly from sorting endosomes, in a process that does not require hydrolysis of GTP on rab11. The fact that the dominant negative (S25N) form of rab11 inhibits recycling of molecules interiorized at both temperatures indicates that activation of rab11 by GTP is required for exit of transferrin from sorting endosomes, regardless of whether this exit is toward the recycling compartment or directly to the plasma membrane.

Figure 1

Localization of rab11 in the pericentriolar recycling compartment and the inhibitory effect of the S25N mutant on the accumulation of FITC–transferrin in that compartment. TRVb-1 cells that express the human transferrin receptor were transfected for transient expression with plasmids encoding: (A and B) the wild type (wt); (C and D) constitutively active (Q70L); (E and F) dominant negative (S25N); or (G and H) C-terminally deleted (ΔC) forms of rab11, as indicated on the left side of each pair of panels. Cells on coverslips were incubated with FITC–transferrin (FITC-Tf) for 1 h at 37°C and processed for indirect immunofluorescence with anti-rab11 antibody (rab11 Ab) and Texas Red-conjugated second antibody. Each pair of photographs (A-H) shows, for the same cells, the distribution of FITC–transferrin (left panels) and Texas Red (right panels) fluorescence. All cells express the transferrin receptor, so they all display intense green fluorescence of FITC–transferrin. The long arrows indicate the pericentriolar recycling compartment in the transfected cells, which in the right panels are identified by their intense Texas Red fluorescence. The short arrows point to the pericentriolar compartment in untransfected cells within the same cultures, and the arrowheads point to cytoplasmic vesicles containing both markers. Note that in cultures transfected with the S25N dominant negative rab11 mutant (E and F), the labeling of the pericentriolar compartment is diminished markedly in transfected cells but not in untransfected cells. Cells expressing the ΔC mutant of rab11 show a diffuse distribution of this protein.

Figure 2

The constitutively active (Q70L) and wild-type rab11, as well as the dominant negative (S25N) form of this protein, inhibit the recycling of transferrin molecules internalized at 37°C. TRVb cells were cotransfected with pairs of plasmids, one encoding the human transferrin receptor and the other encoding a form of rab11, as indicated in each panel (A, Rab11-wt; B, Rab11-Q70L; C, Rab11-S25N; and D, Rab11-ΔC). The transfected cells were incubated with ¹²⁵I-transferrin for 1 h at 37°C, to fully load the various intracellular compartments. The cells then were washed, stripped of surface-bound ligand, and incubated at 37°C in medium containing unlabeled transferrin. The extent of release of the internalized marker into the medium was monitored at various times thereafter. The percentage of ¹²⁵I-transferrin that remains cell-associated in cells expressing each rab11 variant is shown in a semi-log plot as a function of time, together with equivalent data from control cells transfected with the vector lacking a rab11 cDNA insert. Except for rab11-ΔC, the data represent the averages of two separate experiments. The straight lines were obtained by nonlinear curve fitting to the equation y = A_* exp (−K_r ⋅ t), where y is the percent of cell-associated ¹²⁵I-transferrin and K_r the apparent first order recycling rate constant. The rate constants for the different transfected cultures are listed in Table 1.

Figure 3

FITC–transferrin molecules endocytosed at 16°C do not reach the pericentriolar recycling compartment in which rab11 is concentrated. TRVb-1 cells that express the human transferrin receptor were transfected, as in Fig. 1, with the various rab11 plasmids indicated on the left of each pair of panels. The cells grown on coverslips were allowed to take up FITC–transferrin (FITC-Tf) for 1 h at 16°C, before processing, as in Fig. 1, for double immunofluorescence to localize internalized transferrin (left panels) and rab11 (right panels). The long arrows indicate the pericentriolar recycling compartment identified by the high concentration of rab11. Short arrows point to the recycling compartment in untransfected cells within the same cultures, and the arrowheads point to cytoplasmic vesicles containing both markers. Note that in all cases (left panels), FITC–transferrin is distributed in cytoplasmic vesicles and is not concentrated in the pericentriolar compartment, which in B and D is intensely labeled with anti-rab11 antibodies.

Figure 4

The constitutively active (Q70L) and wild-type rab11 do not inhibit recycling at 37°C of ¹²⁵I-transferrin molecules internalized during a previous incubation at 16°C, although the dominant negative form (S25N) of rab11 still inhibits recycling. This experiment was carried out as that in Fig. 3, except that the cells were loaded with ¹²⁵I-transferrin for 1 h by incubation at 16°C rather than at 37°C.