Di-leucine signals mediate targeting of tyrosinase and synaptotagmin to synaptic-like microvesicles within PC12 cells

Abstract

One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRP-tyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents.

Figure 1

Schematic illustration showing wild-type and mutant HRP-tyrosinase chimeras. The top line shows the components used for constructions as follows: hGH, human growth hormone signal; HRP, enzimatically active domain of HRP; P-Sel, the transmembrane domain of P-selectin; and Tyrosinase, the cytoplasmic domain of human tyrosinase. Boxes show the sequence boundaries of individual components. Sequences outside boxes show the components added during construction. The middle line shows the full amino acid sequence of the cytoplasmic tail of tyrosinase as used in wild-type HRP-tyrosinase. The bottom line shows the sequence of the cytoplasmic domain of tyrosinase with di-alanine substitution of di-leucine; HRP-tyrosinase/AA. Amino acid numbers from human tyrosinase are shown.

Figure 2

Targeting of wild-type and mutant HRP-tyrosinase chimeras to SLMV in PC12 cells. (A) PC12 cells expressing either wild-type HRP-tyrosinase or HRP-tyrosinase/AA were homogenized, and PNS was then fractionated on 5–25% glycerol gradients to isolate SLMV. HRP activity for wild-type (●) and for mutant (∗) chimeras is expressed in arbitrary units representing the amount of HRP activity in each fraction divided by that in the homogenate. (B) Cells expressing wild-type HRP-tyrosinase were incubated in the presence (10 μg/ml; ○) or absence (●) of BFA for 1 h at 37°C and processed by subcellular fractionation on glycerol gradients. HRP activity is expressed in arbitrary units as indicated in A. Aliquots from each fraction across the gradient shown in B (●) were separated by 10% SDS-PAGE and Western blotted with polyclonal antibodies against synaptophysin/p38 (C) or against synaptotagmin/p65 (D). The left tracks on both blots represent p38 or p65 in the homogenate.

Figure 3

Schematic illustration showing the CD4-synaptotagmin chimeras. The top line shows the structure of synaptotagmin I, which consists of a lumenal domain (empty box), transmembrane domain (striped box), and cytoplasmic tail comprising C2A, C2B (filled boxes), and the C terminus (gray box). The full amino acid sequence of the C terminus is indicated above. The middle section shows the CD4-synaptotagmin chimeras in which lumenal and transmembrane domains were those from CD4 (empty and dotted boxes, respectively), followed by the entire (wild-type CD4-C2AB) cytoplasmic tail of synaptotagmin I. The position of the di-alanine substitution of Met-Leu within the C terminus is shown in the insert for CD4-C2AB/AA. The lower section illustrates those chimeras in which deletions of the cytoplasmic domain of synaptotagmin have been fused with CD4. CD4-C2A, chimera with truncation both of C2B and the C terminus; CD4-C2B, chimera with a deletion of C2A; CD4-C-terminus, chimera with deletion both of C2A and C2B; CD4-tailless, chimera in which the whole cytoplasmic tail of synaptotagmin has been removed. Positions of di-Ala substitutions within the C terminus are shown within the gray boxes and are reflected in the chimera’s name.

Figure 4

Targeting of CD4-C2AB to SLMV in PC12 cells. PC12 cells expressing wild-type CD4-C2AB were fed with 100 ng/ml ¹²⁵I-Q4120 in the presence (○) or absence (●) of 10 μg/ml BFA and fractionated on 5–25% glycerol gradients to isolate SLMV. The efficiency of SLMV targeting is expressed as the amount of radioactivity in each fraction across the gradient normalized to that in the homogenate.

Figure 5

SLMV targeting of CD4-synaptotagmin chimeras. PC12 cells expressing the chimera indicated were fed with 100 ng/ml ¹²⁵I-Q4120 in the presence or absence of BFA and fractionated on glycerol gradients. The efficiency of targeting to SLMV was calculated as the amount of ¹²⁵I-Q4120 radioactivity within SLMV peak divided by that in the homogenate. (A) Targeting efficiency is expressed on a scale related to the wild-type CD4-C2AB (1) and the CD4-tailless chimera (0). Each bar represents the mean ± SE of five independent experiments. Deviations of <0.015 are not displayed. (B) The targeting efficencies of CD4-C2AB, CD4-C2B, and CD4-C-terminus are expressed such that each individual chimera in the absence of BFA has an efficiency of 1.

Figure 6

The efficiency of SLMV targeting of CD4-synaptotagmin chimeras with di-alanine substitutions. PC12 cells expressing the indicated chimera were incubated with 100 ng/ml ¹²⁵I-Q4120 in the presence or absence of BFA and fractionated to determine SLMV targeting as described in the legend for Figure 4. The efficiency of SLMV targeting for each chimera was calculated as the amount of ¹²⁵I-Q4120 radioactivity within the SLMV peak normalized to that in the homogenate, expressed on a scale where 1 corresponds to the targeting efficiency of wild-type CD4-C2AB in the absence of BFA and 0 corresponds to that of CD4-tailless. Each bar represents the mean ± SE of three independent experiments. Deviations of <0.015 are not displayed.

Figure 7

Endocytosis of ¹²⁵I-Q4120 in PC12 cells expressing CD4-synaptotagmin chimeras. Cells expressing CD4-C2AB (●), CD4-C2AB/AA (○), CD4-C2B (▴), CD4-C2B/AA (▵), CD4-C-terminus (▪), CD4-C-terminus/AA (□), or CD4-tailless (X) were incubated with 100 ng/ml ¹²⁵I-Q4120 at 4°C for 1 h and allowed to internalize the ligand for 0, 5, 10, or 15 min at 37°C. Antibodies remaining on the cell surface were then removed by washing with acetic buffer at 4°C. Intracellular radioactivity was normalized to the total radioactivity bound to the cells and expressed as percentages.