The clathrin adaptor complex 1 directly binds to a sorting signal in Ste13p to reduce the rate of its trafficking to the late endosome of yeast

Abstract

Yeast trans-Golgi network (TGN) membrane proteins maintain steady-state localization by constantly cycling to and from endosomes. In this study, we examined the trafficking itinerary and molecular requirements for delivery of a model TGN protein A(F-->A)-alkaline phosphatase (ALP) to the prevacuolar/endosomal compartment (PVC). A(F-->A)-ALP was found to reach the PVC via early endosomes (EEs) with a half-time of approximately 60 min. Delivery of A(F-->A)-ALP to the PVC was not dependent on either the GGA or adaptor protein 1 (AP-1) type of clathrin adaptors, which are thought to function in TGN to PVC and TGN to EE transport, respectively. Surprisingly, in cells lacking the function of both GGA and AP-1 adaptors, A(F-->A)-ALP transport to the PVC was dramatically accelerated. A 12-residue cytosolic domain motif of A(F-->A)-ALP was found to mediate direct binding to AP-1 and was sufficient to slow TGN-->EE-->PVC trafficking. These results suggest a model in which this novel sorting signal targets A(F-->A)-ALP into clathrin/AP-1 vesicles at the EE for retrieval back to the TGN.

Figure 1.

GFP-tagged A-ALP partially colocalizes with EEs. Strains SHY35/pCF17 and SHY35/pG-P12-U were stained at 0°C with FM4-64, and the dye was allowed to internalize by incubating in media at 30°C for 2 or 8 min followed by the addition of NaN₃ and NaF to halt traffic. (A) After 2 min of internalization, GFP-ALP, GFP-Pep12p, and FM4-64 were imaged as indicated. (B) The percentage of punctate FM4-64 structures that were positive for either GFP–A-ALP or GFP-Pep12p was quantified after 2 and 8 min of incubation at 30°C. A minimum of 150 punctate structures were analyzed for each data point.

Figure 2.

A(F→A)-ALP uses a TGN→EE→PVC pathway before reaching the vacuole. Wild-type (SHY35), soi3Δ (CFY38), vps8Δ (CFY37), pep 12-49 ^ts (SNY156), gga1,2Δ (SNY165), and gga1,2Δ end3-ts (SNY171-4D) strains carrying a plasmid expressing A(F→A)-ALP (pSN100) were analyzed. Cells were pulsed for 10 min with [³⁵S]methionine/cysteine and chased for the indicated times. The strains were either incubated at 30°C throughout the time course (A) or were propagated for several doublings at 24°C before shifting to 36°C for 10 min before initiation of the chase (B and C). After each time point, A(F→A)-ALP was immunoprecipitated and analyzed by SDS-PAGE to separate the precursor (p) and mature (m) forms. The half-time of processing of each strain is indicated below the panels in A and C.

Figure 3.

Cps1p uses a GGA-dependent direct TGN to PVC pathway before reaching the vacuole. Strains CFY30, CFY32, CFY33, and CFY31 (from left to right) carrying a CEN-CPS1 plasmid were analyzed in A, whereas strains SHY35, CFY38, and CFY37 were analyzed in B. The strains were either propagated for several doublings at 24°C before shifting to 36°C for 10 min before the initiation of the chase (A) or were incubated at 30°C throughout the time course (B). After each chase time, Cps1p was immunoprecipitated, treated with endoglycosidase H, and analyzed by SDS-PAGE to separate the precursor (p) and mature (m) forms. The half-time of processing of each strain is indicated below each panel.

Figure 4.

Both the adaptor complex AP-1 and the Ste13p 2–11 region slow the transport of A(F→A)-ALP through the TGN→EE→PVC pathway rather than being required for anterograde transport. (A) Strains SHY35 (wild type), UFY2 (apl2Δ), SNY165 (gga1,2Δ), CFY6-2C/pCF2 (apl2Δ gga1,2Δ /pCEN-APL2), and CFY6-2C/pCF6 (apl2Δ gga1,2Δ /pCEN-apl2-ts) were analyzed by spotting 10-fold serial dilutions onto YPD media and incubating for 4 d at the indicated temperature. (B) SHY35, CFY6-2C/pCF6, and CFY25-3B/pCF6 (apl2Δ gga1,2Δ end3-ts/pCEN-apl2-ts) strains were grown for several doublings at 24°C, shifted to 36°C (or left at 24°C as indicated) for 10 min, pulsed for 10 min, and chased as indicated. (C) Strain CFY6-2C/pCF6 carrying (left to right) plasmids pSN100 (A(F→A)-ALP), pHJ63 (A(S₁₃A; F→A)-ALP), or pSH46 (Δ2–11; F→A)-ALP) was analyzed after shifting from 24 to 36°C as in B. (D) Strains SHY35, SNY94 (end3-ts), SNY165, and SNY171-4D carrying pSN100 (top) or pSH46 (bottom) were analyzed after shifting from 24 to 36°C as in B. (B–D) A(F→A)-ALP or its derivatives were immunoprecipitated and analyzed by SDS-PAGE to separate the precursor (p) and mature (m) forms. The half-time of processing of each strain is indicated below each panel.

Figure 5.

The clathrin adaptor complex AP-1 directly interacts with amino acids 1–12 of Ste13p. The following proteins were expressed in E. coli and purified onto glutathione-agarose beads: Ste13-GST (WT), Ste13(S₁₃A)-GST (S₁₃A), Ste13(S₁₃D)-GST (S₁₃D), Ste13(1–20)-GST (1-20), Ste13 (1–12)-GST (1-12), and GST (Vect or −). (A) The bead samples were incubated with an SNY190 yeast protein extract followed by washing, elution, and analysis of the eluted proteins by SDS-PAGE. Identical gels were immunoblotted with anti-Apl2p or anti-HA antibodies to detect Apm1-HA as indicated. Yeast extract equivalent to 4% of that incubated with each bead sample was also analyzed (input). The same samples analyzed by Western blotting were also analyzed by staining with Coomassie brilliant blue (CBB) to indicate the relative quantity of each GST fusion. (B) Rabbit reticulocyte lysates programmed with mRNA encoding either Apm1p (left) or an Apm1-Δ2–158 mutant (right) were used to synthesize the corresponding ³⁵S-labeled proteins. Beads prebound to the indicated Ste13-GST constructs were incubated with aliquots of each in vitro translation reaction. After washing, elution, and separation by SDS-PAGE, bead-associated proteins were analyzed by Coomassie staining (bottom) followed by autoradiography (top). The input represents 10% of the translation reaction incubated with each of the bead samples. (C) The following maltose-binding protein (MBP)–derived proteins were expressed in E. coli, purified onto amylose resin, and eluted using maltose: MBP-Apm1, MBP–Apm1-Δ2–158, and MBP alone. Beads prebound to Ste13-GST and GST alone were incubated with each MBP-derived protein. Aliquots of proteins not associated with beads (unbound) and proteins that remained associated with beads after washing (bound) were separated by SDS-PAGE and subjected to anti-MBP immunoblotting. Bead-associated samples were also analyzed by Coomassie brilliant blue staining to visualize the GST-derived proteins. For each of the three panels, samples were loaded on the same gel, proteins were detected, and lanes were rearranged for presentation. A total of 60 and 3% of the bound and unbound samples were loaded, respectively, on the indicated gels that were subsequently processed into immunoblots using identical conditions to facilitate comparison. The positions and size (in kilodaltons) of molecular mass standards are indicated.

Figure 6.

Residues 1–12 of Ste13p are sufficient to slow the trafficking of Cps1p into the PVC/vacuole. Yeast strains CFY30 (cps1Δ) and CFY32 (cps1Δ gga1,2Δ) carrying plasmids expressing Cps1p, Ste13(1–23)-Cps1, Ste13(1–23; S₁₃A)-Cps1, and Ste13(1–12)-Cps1 were subjected to pulse-chase analysis at 30°C and immunoprecipitation as described in Fig. 3. The half-time of processing is indicated below each panel. The gga1,2Δ half-times are provided as the average and SD of two independent datasets. The positions and size (in kilodaltons) of molecular mass standards are indicated. p, precursor form; m, mature form.

Figure 7.

Models for the role of clathrin/AP-1 in the sorting of A(F→A)-ALP. (A) A(F→A)-ALP undergoes repeated rounds of cycling between the TGN and EE that involve sorting into clathrin/AP-1–coated vesicles at the EE. (B) Clathrin/AP-1 mediates the static retention of A(F→A)-ALP at the TGN. In both models, sorting is mediated by the interaction of AP-1 with residues 1–12 of A(F→A)-ALP. The GGA clathrin adaptors may participate with AP-1 in sorting A(F→A)-ALP.