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. 2000 May;11(5):1801-14.
doi: 10.1091/mbc.11.5.1801.

Sorting to synaptic-like microvesicles from early and late endosomes requires overlapping but not identical targeting signals

A D Blagoveshchenskaya  1 , D F Cutler
Affiliations
Free PMC article

Sorting to synaptic-like microvesicles from early and late endosomes requires overlapping but not identical targeting signals

A D Blagoveshchenskaya et al. Mol Biol Cell. 2000 May.
Free PMC article

Abstract

In PC12 neuroendocrine cells, synaptic-like microvesicles (SLMV) are thought to be formed by two pathways. One pathway sorts the proteins to SLMV directly from the plasma membrane (or a specialized domain thereof) in an adaptor protein complex 2-dependent, brefeldin A (BFA)-insensitive manner. Another pathway operates via an endosomal intermediate, involves adaptor protein complex 3, and is BFA sensitive. We have previously shown that when expressed in PC12 cells, HRP-P-selectin chimeras are directed to SLMV mostly via the endosomal, BFA-sensitive route. We have now found that two endosomal intermediates are involved in targeting of HRP-P-selectin chimeras to SLMV. The first intermediate is the early, transferrin-positive, epidermal growth factor-positive endosome, from which exit to SLMV is controlled by the targeting determinants YGVF and KCPL, located within the cytoplasmic domain of P-selectin. The second intermediate is the late, transferrin-negative, epidermal growth factor-positive late endosome, from where HRP-P-selectin chimeras are sorted to SLMV in a YGVF- and DPSP-dependent manner. Both sorting steps, early endosomes to SLMV and late endosomes to SLMV, are affected by BFA. In addition, analysis of double mutants with alanine substitutions of KCPL and YGVF or KCPL and DPSP indicated that chimeras pass sequentially through these intermediates en route both to lysosomes and to SLMV. We conclude that a third site of formation for SLMV, the late endosomes, exists in PC12 cells.

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Figures

Figure 1
Figure 1
Schematic illustration of HRP-P-selectin chimeras and localization of major targeting determinants within the cytoplasmic tail of P-selectin. (A) The top line shows the position of components used for construction: hGH, human growth hormone signal sequence; P-selectin, transmembrane and cytoplasmic domain of P-selectin. The cytoplasmic domain of P-selectin was divided into the stop transfer (ST), C1, and C2 subdomains according to exon–intron boundaries (Johnston et al., 1989). The bottom part shows the full amino acid sequences of the cytoplasmic domains of the chimeras. The carboxyl-terminal end of the transmembrane domain shown is boxed. The amino acids substituted for alanine are shown to the left of the diagram and included in name of the chimera. ssHRPP-selectin763 is a chimera in which both the C1 and C2 subdomains are removed. (B) Localization of SLMV and lysosomal targeting signals within the cytoplasmic domain of P-selectin. The determinants inactivation of which reduces targeting to the level of tailless ssHRPP-selectin763, are shown within the + boxes. The determinants inactivation of which increases targeting over the level of wild-type ssHRPP-selectin, are shown within the − boxes.
Figure 2
Figure 2
Quantitation of targeting of HRP-P-selectin chimeras to early endosomes. PC12 cells expressing the chimera indicated were labeled with 125I-Trn, treated with ascorbate to inhibit HRP activity present on the plasma membrane, homogenized, and fractionated using 1–16% Ficoll velocity gradients followed by 3–16% Ficoll velocity gradients to isolate early endosomes. After fractionation, targeting to early endosomes was measured by calculating a targeting index (EE-TI) for each chimera as the amount of HRP activity within the peak of early endosomes normalized both by the expression level and by 125I-Trn recovery. Each bar represents the mean ± SE of three independent experiments.
Figure 3
Figure 3
Quantitation of targeting of HRP-P-selectin chimeras to late endosomes. PC12 cells expressing the chimeras indicated were homogenized and fractionated on 1–16% Ficoll velocity gradients followed by recentrifugation on 0.9–1.85 M sucrose equilibrium gradients for separation of late endosomes. LE-TIs were then calculated by normalizing the amount of HRP activity within the endosomal peak both for organelle recovery as judged by NAGA activity and for the expression level. Each bar represents the mean ± SE of three independent experiments.
Figure 4
Figure 4
Effect of different levels of expression on targeting of HRP-P-selectin chimeras to late endosomes. (A) Titration of HRP expression. PC12 cells were transiently transfected by electroporation using increasing amounts of cDNA for ssHRPP-selectin (WT), ssHRPP-selectinKCPL (KCPL), or ssHRPP-selectinDPSP (DPSP). Cells were washed with HB, scraped, and homogenized. HRP activity (shown in arbitrary units [A.U.]) was measured in each homogenate and normalized to total protein within the homogenate. The name of chimera and the amount of DNA used (starting from 0.5 μg) are shown along the abscissa. (B) Efficiency of late endosomal targeting at different expression levels. PC12 cells transiently expressing ssHRPP-selectin, ssHRPP-selectinKCPL, or ssHRPP-selectinDPSP at different levels were processed by subcellular fractionation for isolation of late endosomes as described in the legend for Figure 3. The amount of HRP activity (shown in A.U. along the ordinate) within the late endosomal peak was measured, divided by that in the homogenate, and normalized for NAGA recovery. The amount of DNA used is shown before the name of the chimera. Note that the level of constitutive targeting of tailless ssHRPP-selectin763 to late endosomes, which usually comprises 25% of the level found for the wild-type chimera, has not been subtracted from the values of targeting efficiency for chimeras analyzed in this experiment.
Figure 5
Figure 5
Immunofluorescent localization of internalized HRP-P-selectin chimeras and endosomal markers in PC12 cells. Cells transiently expressing wild-type ssHRPP-selectin (A–C), tailless ssHRPP-selectin763 (D–F), ssHRPP-selectinDPSP (G–I), or ssHRPP-selectinKCPL (J–O) grown on poly-l-lysine-coated coverslips were washed with serum-free medium containing 1% BSA and fed with 50 μg/ml Trn (J–O) and/or with 5 μg/ml 2H11 (A–O) for 2 h at 37°C. Cells were then fixed, permeabilized, and stained as described in MATERIALS AND METHODS. 2H11 (A, D, G, J, and M) was visualized with Texas Red-conjugated goat anti-mouse secondary antibody; LAMP1 (B, E, H, and K) was immunodetected with rabbit polyclonal anti-LAMP1 followed by FITC-conjugated goat anti-rabbit secondary antibody; and Trn (N) was detected with rabbit polyclonal anti-Trn followed by FITC-conjugated goat anti-rabbit secondary antibody. Color images show the merger of both channels.
Figure 6
Figure 6
Compartmentalization of internalized 125I-Trn and 125I-EGF in the presence or absence of BFA. PC12 cells expressing wild-type ssHRPP-selectin were fed with 125I-Trn for 1 h at 37°C in the presence (●) or absence (○) of 10 μg/ml BFA added in the medium during last 30 min of incubation. Parallel dishes were incubated with 125I-EGF for 1 h on ice in the presence or absence of BFA for the last 10 min of incubation, washed, and transferred to 37°C for 20 min to label late endosomes in the presence (▴) or absence (▵) of BFA. One dish labeled with 125I-EGF for 1 h on ice was washed and allowed to internalize ligand for 2 min at 37°C with no BFA added (A, ∗). After removal of the noninternalized ligand, cells were rinsed with HB and homogenized, and a PNS was centrifuged on the 1–16% Ficoll velocity gradients (A and B). After fractionation, the peak containing 125I-Trn (fractions 5–10; A) was collected and then recentrifuged on a 3–16% Ficoll gradient (C), whereas the peak containing 125I-EGF (fractions 13–19; B) was collected and recentrifuged on a 0.9–1.85 M sucrose equilibrium gradient (D) as described in MATERIALS AND METHODS.
Figure 7
Figure 7
Effect of BFA on the distribution of wild-type ssHRPP-selectin along the 1–16% Ficoll initial gradients and along the secondary gradients used for isolation of early and late endosomes. PC12 cells expressing wild-type ssHRPP-selectin were fed with 125I-Trn or with 125I-EGF to monitor the position of early or late endosomes and incubated in the presence (▪) or absence (□) of BFA as described in the legend for Figure 6. After centrifugation and fractionation on 1–16% Ficoll gradients, HRP activity was determined in each fraction and divided by that in the homogenate (A). Fractions corresponding to early endosomes (5–10), as seen by distribution of 125I-Trn (Figure 6A), were pooled together and recentrifuged on 3–16% Ficoll gradients (B). Fractions corresponding to late endosomes (13–19), as judged by distribution of 125I-EGF internalized for 20 min at 37°C (Figure 6B), were collected and recentrifuged on 0.9–1.85 M sucrose equilibrium gradients (C). After fractionation, HRP activity was measured in each fraction and normalized to that in the homogenate.
Figure 8
Figure 8
Effect of BFA on the distribution of ssHRPP-selectinYGVF and ssHRPP-selectinKCPL along the endocytic pathway. PC12 cells expressing either ssHRPP-selectinYGVF (A–C) or ssHRPP-selectinKCPL (D and E) were incubated in the presence (filled symbols) or absence (open symbols) of BFA, homogenized, and fractionated on the 1–16% Ficoll gradients. Fractions corresponding to early or late endosomes for ssHRPP-selectinYGVF were collected separately and recentrifuged on 3–16% Ficoll gradients (B) or 0.9–1.85 M sucrose gradients (C), respectively. The peak of early endosomes for ssHRPP-selectinKCPL was pooled (D) and recentrifuged on a 3–16% Ficoll gradient (E). In all cases, HRP activity was measured in each fraction across the gradient and divided by that in the homogenate. One representative experiment of two is shown.
Figure 9
Figure 9
Schematic model for trafficking of HRP-P-selectin chimeras to SLMV in PC12 cells. HRP-P-selectin present on the plasma membrane is internalized into early Trn-positive, EGF-positive endosomes (EE) from which budding of SLMV occurs in a KCPL- and YGVF-dependent and BFA-sensitive manner. From the same endosomes, the remaining chimera is sorted to late, Trn-negative, EGF-positive endosomes (LE) in a KCPL-dependent, BFA-insensitive manner. From this compartment, further trafficking of P-selectin can occur in one of the two directions. One route leads to SLMV, a process that is controlled by YGVF and DPSP and is BFA sensitive. The remaining protein within LE is delivered to lysosomes (Lys) for degradation. Another potential SLMV precusor, represented by invaginations of the plasma membrane, from which budding of SLMV is BFA insensitive (12% for HRP-P-selectin vs. 88% that is BFA sensitive) and which does not involve an endosomal intermediate, is shown in the inset.
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