UNC-18 promotes both the anterograde trafficking and synaptic function of syntaxin

Abstract

The SM protein UNC-18 has been proposed to regulate several aspects of secretion, including synaptic vesicle docking, priming, and fusion. Here, we show that UNC-18 has a chaperone function in neurons, promoting anterograde transport of the plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein Syntaxin-1. In unc-18 mutants, UNC-64 (Caenorhabditis elegans Syntaxin-1) accumulates in neuronal cell bodies. Colocalization studies and analysis of carbohydrate modifications both suggest that this accumulation occurs in the endoplasmic reticulum. This trafficking defect is specific for UNC-64 Syntaxin-1, because 14 other SNARE proteins and two active zone markers were unaffected. UNC-18 binds to Syntaxin through at least two mechanisms: binding to closed Syntaxin, or to the N terminus of Syntaxin. It is unclear which of these binding modes mediates UNC-18 function in neurons. The chaperone function of UNC-18 was eliminated in double mutants predicted to disrupt both modes of Syntaxin binding, but it was unaffected in single mutants. By contrast, mutations predicted to disrupt UNC-18 binding to the N terminus of Syntaxin caused significant defects in locomotion behavior and responsiveness to cholinesterase inhibitors. Collectively, these results demonstrate the UNC-18 acts as a molecular chaperone for Syntaxin transport in neurons and that the two modes of UNC-18 binding to Syntaxin are involved in different aspects of UNC-18 function.

Figure 1.

Syntaxin-1 accumulates in cell bodies of unc-18 mutants. Animals were Bouin's fixed and stained with anti-UNC-64 Sytaxin-1 antibodies. (A) In wild-type animals, all neuronal processes are labeled and a small number of cell bodies can be seen. In unc-18(md299) and unc-18(e81) mutants, cell bodies accumulate UNC-64 brightly and can be easily visualized. Other synaptic mutants, unc-10(e102) and snb-1(md247) have similar staining patterns as wild type. (B) Quantification of the number of visible cell bodies along the ventral nerve cord of animals (n = 20 for each genotype). (C) GFP::UNC-64 Syntaxin-1 expressed under the UNC-129 promoter in the DA motor neurons. In wild-type animals, GFP::UNC-64 is diffuse. In unc-18(md299) GFP::UNC-64 accumulates in cell bodies. Bars, 10 μm. Values that differ significantly from wild type based on the Student's t test, **p < 0.001). Error bars represent SEM.

Figure 2.

Localization of other presynaptic SNAREs is UNC-18 independent. Wild-type and unc-18(md299) animals were Bouin's fixed and stained with antibodies against SNB-1 Syntaptobrevin-1 (A) and RIC-4 SNAP-25 (B). The distribution of both SNARE proteins was unaffected in unc-18 (md299) animals. Bars, 10 μm.

Figure 3.

Most C. elegans SNARE proteins are trafficked normally in unc-18 mutants. SNARE proteins were tagged with either GFP or RFP and then expressed under the pan-neuronal snb-1 promoter. Fluorescently tagged SNARE proteins were then compared in wild-type versus unc-18(md299) mutant animals. There was no obvious trafficking defect for the 12 SNAREs analyzed in unc-18 mutants. Bar, 10 μm.

Figure 4.

Active zone proteins αLiprin and RIM1 are localized normally in unc-18 mutants. GFP tagged SYD-2 αLiprin and UNC-10 RIM1 were expressed under the UNC-129 promoter in a subset of motor neurons. (A) SYD-2 αLiprin::GFP is localized to presynaptic active zones along the dorsal nerve cord and is not changed in unc-18 (md299) compared with wild-type animals. (B) UNC-10 RIM1::GFP has a similar presynaptic distribution and is not changed in unc-18 (md299) compared with wild-type animals. Bars, 10 μm.

Figure 5.

An N-Glycosylated Syntaxin-1 accumulates in the ER in unc-18 mutants. (A) Lysates of GFP::UNC-64::5N expressing worms were untreated or treated with either Endo H or PNGase F and analyzed by Western blot with anti-UNC-64 Syntaxin-1 antibodies. In all untreated lysates, GFP::UNC-64::5N occurs as a series of bands ranging from 80 to 100 kDa. PNGase treatment results in a single band near 80 kDa, demonstrating efficient N-glycosylation of GFP::UNC-64::5N. In unc-18(md299) animals, there is an increase in the amount of GFP::UNC-64::5N that is sensitive to the enzyme Endo H, indicating a larger fraction of GFP::UNC-64::5N present in pre-Golgi compartments compared with wild type and ric-4(md1088) controls. (B) Summary data of quantitative Western blots for each genotype indicated (n = 5 per genotype) shows that in unc-18 mutants there is a twofold increase in the Endo H-sensitive fraction of GFP::UNC-64::5N. (C and D) GFP::UNC-64 was expressed under the UNC-129 promoter in a subset of motor neurons along with ssRFP::KDEL to determine colocalization with the ER compartment. (C) GFP::UNC-64 expressed in wild-type animals is mostly localized to the cell periphery separate from ssRFP::KDEL. (D) In unc-18(md299) mutant animals, GFP::UNC-64 has increased colocalization with ssRFP::KDEL and decreased abundance at the cell periphery. Bars, 2 μm. (E) Quantification of colocalization of GFP::UNC-64 and the amount of overlap with ssRFP::KDEL is shown (n = 10). (F) Quantification of GFP::UNC-64 fluorescence levels along the dorsal nerve cord is shown (n = 10). (G and H) GFP::UNC-64 imaged along the dorsal nerve cord. (G) Mostly diffuse GFP::UNC-64 is seen along the nerve cord in wild-type animals. (H) In unc-18(md299) animals, GFP::UNC-64 fluorescence is decreased along the nerve cord. Bar, 10 μm. Values that differ significantly from wild type, *p < 0.01, Student's t test). Values that differ significantly from wild-type animals, **p < 0.001, Student's t test. Error bars represent SEM.

Figure 6.

Rescue of endogenous UNC-64 trafficking with UNC-18::FLAG and UNC-18::FLAG mutants. Fixed animals were immunostained with anti-UNC-64 Syntaxin-1 antibodies (Red). (A) Wild-type animals stained with anti-UNC-64 show very few visible cell bodies. (B) unc-18(md299) mutant animals show an accumulation of UNC-64 in the cell bodies. (C–H) unc-18(md299) mutant animals carrying a transgenic array expressing either wild-type UNC-18::FLAG or point mutants. (C′–H′) Anti-FLAG staining to show expression of UNC-18::FLAG constructs (Green). UNC-64 transport was rescued by transgenes expressing wild-type UNC-18 (C), UNC-18(R39C) (D), UNC-18(D34N) (E), and UNC-18(L116K) (F). Rescue was not observed with transgenes expressing the double mutants UNC-18(R39C; L116K) (G), and UNC-18(D34N; L116) (H). Bars, 10 μm.

Figure 7.

Effect of UNC-64 mutations on anterograde trafficking. (A–E) GFP::UNC-64 variants and an ER marker (ssRFP::KDEL) were expressed in the cholinergic DA motor neurons (using the unc-129 promoter). Wild-type GFP::UNC-64 is primarily found at the cell periphery in wild-type neurons (A), but they had increased colocalization with ssRFP::KDEL and decreased abundance at the cell periphery in unc-18(md299) mutants (B). A wild-type distribution was observed for UNC-64(open) (C) and UNC-64(L9A) (D), whereas increased colocalization with ssRFP::KDEL was observed for UNC-64(open; L9A) (E). (F) Quantification of colocalization of GFP::UNC-64 variants with ssRFP::KDEL is shown (n = 10). The wild-type GFP::UNC-64 data in wild-type and unc-18(md299) mutants are the same as shown in Figure 5E. Bars, 2 μm. Values that differ significantly from GFP::UNC-64 in wild-type controls based on the Student's t test, **p < 0.001. Error bars represent SEM.

Figure 8.

Mutations that disrupt UNC-18 binding to the N terminus of UNC-64 have behavioral defects. (A–E) UNC-18 constructs were expressed in unc-18(md299) mutants, UNC-64 constructs were expressed in the unc-64(js115) background as indicated. (A–C) Animals were placed on NGM plates containing 1 mM aldicarb and assayed over time for percentage of paralysis. The percentage of animals paralyzed at 140 min (A and B) and 50 min (C) are summarized. (D and E) The number of body bends per 30 s on NGM plates without food as a measure of locomotion for indicated transgenes expressed in unc-18(md299) mutants (D) and unc-64(js115) mutants (E). R39C, D34N, and L116K are indicators for UNC-18::FLAG transgenes containing the indicated point mutations in the UNC-18 protein. L9A and Open represent UNC-64 transgenes containing the indicated point mutations in the UNC-64a protein. Values that differ significantly from animals rescued with wild-type constructs based on the Student's t test, **p < 0.001). Error bars represent SEM. Daggers (†) indicates use of the oxIs34 [open-UNC-64] strain that does not contain N-terminal GFP.