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Vol. 17, Issue 10, 4282-4299, October 2006

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Identification of the Yeast R-SNARE Nyv1p as a Novel Longin Domain-containing Protein

Wenyu Wen^*,, Lu Chen^,, Hao Wu^*, Xin Sun, Mingjie Zhang^*, and David K. Banfield

Departments of *Biochemistry and Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, Special Administrative Region of China

Submitted February 13, 2006; Revised June 20, 2006; Accepted July 11, 2006
Monitoring Editor: Benjamin Glick

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Using nuclear magnetic resonance spectroscopy, we establish that the N-terminal domain of the yeast vacuolar R-SNARE Nyv1p adopts a longin-like fold similar to those of Sec22b and Ykt6p. Nyv1p is sorted to the limiting membrane of the vacuole via the adaptor protein (AP)3 adaptin pathway, and we show that its longin domain is sufficient to direct transport to this location. In contrast, we found that the longin domains of Sec22p and Ykt6p were not sufficient to direct their localization. A YXX-like adaptin-dependent sorting signal (Y³¹GTI³⁴) unique to the longin domain of Nyv1p mediates interactions with the AP3 complex in vivo and in vitro. We show that amino acid substitutions to Y³¹GTI³⁴ (Y31Q;I34Q) resulted in mislocalization of Nyv1p as well as reduced binding of the mutant protein to the AP3 complex. Although the sorting of Nyv1p to the limiting membrane of the vacuole is dependent upon the Y³¹GTI³⁴ motif, and Y31 in particular, our findings with structure-based amino acid substitutions in the mu chain (Apm3p) of yeast AP3 suggest a mechanistically distinct role for this subunit in the recognition of YXX-like sorting signals.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The endomembrane system of eukaryotic cells is composed of a complex network of membrane-enclosed organelles, each of which contains a characteristic repertoire of lipid and protein constituents. The functional and compositional integrity of each organelle is maintained by a continued addition to and selective removal of proteins from these compartments, in a process driven by the selectivity of transport step-specific coat protein complexes and mediated by membrane-bound vesicular carriers (Rothman, 1994). Vesicle coat proteins contain binding sites, which recognize sorting motifs on their select protein cargos and direct the recruitment of these proteins into nascent transport vesicles (Lee et al., 2004; Owen et al., 2004). Subsets of vesicle cargo proteins, the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), are required for successful targeting and fusion of transport vesicles with their target organelles (Jahn et al., 2003). In general, SNARE proteins are differentially localized in cells, and trafficking between endomembrane subcompartments requires specific combinations of SNARE proteins. Presumably then, for a transport vesicle to be recognized by and fuse with its target organelle, the vesicle must contain the correct complement of SNARE proteins. Such a requirement implies that vesicle coat protein complexes should be able to selectively recruit the correct repertoire of SNAREs into newly forming transport vesicles. Recent experimental evidence in yeast suggests that this is indeed the case. The COPII coat, which is required for the formation of transport vesicles that deliver proteins from the endoplasmic reticulum (ER) to the Golgi apparatus, contains binding sites for the SNAREs Sed5p, Bet1p, Bos1p, and Sec22p—proteins required for fusion of ER-derived vesicles with the Golgi. Similarly, the vacuolar SNARE Vam3p contains a sorting signal that is required for its sorting to the limiting membrane of the vacuole by the coat/adaptor protein (AP) complex—AP3 (Darsow et al., 1998)—and the endosomal SNARE Pep12p contains a motif required for its sorting from the Golgi to prevacuolar endosomes, in a process presumably mediated by the GGA proteins (Black and Pelham, 2000). In addition, some SNARE proteins contain sorting signals that direct recognition by more than one coat protein complex. For example, the yeast Golgi syntaxin Sed5p cycles between the ER and Golgi (Wooding and Pelham, 1998); thus, presumably, in addition to binding to the COPII coat protein complex, Sed5p also contains a COPI-dependent sorting signal that directs its retrieval from the Golgi back to the ER.

SNARE-mediated fusion typically results in the formation a complex consisting of three Q-SNAREs and one R-SNARE. The Q/R-SNARE nomenclature is based on the x-ray structures of SNARE complexes and denotes whether a conserved glutamine (in Q-SNAREs) or arginine (in R-SNAREs) is located in the so-called zero layer of the complex (Sutton et al., 1998; Antonin et al., 2002). The genome of the yeast Saccharomyces cerevisiae encodes five R-SNAREs: Snc1/2p, Ykt6p, Sec22p, and Nyv1p. Of these five R-SNAREs, Sec22p, Ytk6p, and Nyv1p have N-terminal extensions of their SNARE-motifs greater than 100 amino acids. The structures of the N-terminal domains of Sec22b (the orthologue of yeast Sec22p) and Ytk6p have revealed that both proteins share a similar profilin-like fold (Gonzalez et al., 2001; Tochio et al., 2001). R-SNAREs that contain a profilin-like fold in their N-terminal domains have been termed longins (Filippini et al., 2001; Rossi et al., 2004), and we have adopted this nomenclature here. The longin domain (LD) is not restricted to R-SNAREs and has been identified in a variety of proteins (Rossi et al., 2004), some of which are also involved in vesicular transport. These include subunits of the adaptin complexes (Collins et al., 2002; Heldwein et al., 2004) and SEDL/Trs20p (Jang et al., 2002), a common subunit of the transport protein particle (TRAPP)I and TRAPPII multisubunit complexes, which are required for traffic between the ER and Golgi and within the Golgi (Oka and Krieger, 2005). The function of the longin fold of Sec22b/Sec22p is unknown, but in Ykt6p the longin domain folds back and binds to the SNARE-motif of the protein (Tochio et al., 2001), and this conformation is likely to be important for chaperoning the lipid modified C terminus of the cytoplasmic form of Ykt6p in cells as well as for its localization (Fukasawa et al., 2004; Hasegawa et al., 2004).

Although there is no significant amino acid similarity between the N-terminal domain of Nyv1p and the Ykt6p and Sec22b N termini, structure prediction suggests that the fold of the N-terminal domain of Nyv1p should also resemble those of Sec22p and Ykt6p. Here, we present the structure of the N-terminal domain of Nyv1p, which reveals that Nyv1p is indeed a longin SNARE, and we establish that the longin domain is chiefly responsible for the AP3-dependent sorting of Nyv1p to the limiting membrane of the vacuole via a YXX-like adaptin binding motif.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Expression and Purification of Proteins for Nuclear Magnetic Resonance (NMR) Studies
DNA fragments corresponding to the 149 residue NH₂-terminal domain of Nyv1p (Nyv1p-LD) and its amino acid substitution mutants (nyv1p-LD^Y31Q;I34Q and nyv1p-LD^L73Q;I74Q) and to the 230 residue Nyv1pTMD (Nyv1p minus its transmembrane domain) were amplified by the polymerase chain reaction (PCR). DNA fragments were cloned into a modified pET vector, pETH (Feng et al., 2002) to generate an in-frame fusion of a hexa-histidine [(His)₆]-tag to the C terminus of the proteins. Protein expression was induced in Escherichia coli BL21(DE3) cells by the addition of isopropyl--D-thiogalactopyranoside (IPTG), and the recombinant proteins were purified on Ni²⁺-nitrilotriacetic acid (NTA) affinity columns. After affinity purification, the (His)₆-tag was removed by digesting the fusion proteins with thrombin. Purification of untagged Nyv1p proteins was accomplished by a second Ni²⁺-NTA affinity column purification step, followed by gel filtration chromatography. Uniformly ¹⁵N- and ¹⁵N/¹³C-labeled Nyv1p proteins were prepared by growing bacteria in M9 minimal medium containing ¹⁵NH₄Cl (1 g/l) with or without ¹³C₆-glucose (1 g/l).

NMR Experiments
Four NMR samples were prepared for structural determination of Nyv1p-LD with a protein concentration of 1.0 mM (¹⁵N-labeled protein in 90% H₂O/10% D₂O, two ¹⁵N/¹³C-labeled samples: one sample in 99.9% D₂O and one sample in 90% H₂O/10% D₂O and unlabeled protein in 99.9% D₂O). Protein samples were dissolved in 20 mM potassium phosphate buffer, pH 7.0, containing 6 mM d₁₀-dithiothrietol.

All NMR experiments were carried out at 35°C using Varian Inova 500- and 750-MHz spectrometers. NMR spectra were processed with the nmrPipe software package (Delaglio et al., 1995) and analyzed using PIPP (Garrett et al., 1991) and Sparky (www.cgl.ucsf.edu/home/sparky/). Sequential backbone resonance assignments of the proteins were obtained by standard heteronuclear correlation experiments, including HNCO, HNCACB, and CBCA(CO)NH, and confirmed by a three-dimensional ¹⁵N-separated nuclear Overhauser effect spectroscopy (NOESY) experiments (Kay and Gardner, 1997). The nonaromatic, nonexchangeable side chain assignments were obtained using HCCH-total correlation spectroscopy (TOCSY) experiments. The side chains of aromatics were assigned by ¹H two-dimensional (2D) TOCSY/NOESY experiments with an unlabeled protein sample in D₂O. The stereospecific assignment of the Val and Ile methyl groups was obtained by using a 10% ¹³C-labeled sample (Neri et al., 1989). The -NH₂ side chains of Asn and Gln residues were assigned by a three-dimensional ¹⁵N-separated NOESY experiment in which the ¹⁵N-labeled protein was dissolved in H₂O.

NMR Structural Calculations
Approximate interproton distance restraints were derived from NOESY spectra (a ¹H 2D homonuclear NOESY, a ¹⁵N-separated NOESY, and a ¹³C-separated NOESY, each with a mixing time of 100 ms). Nuclear Overhauser effects (NOEs) were grouped into three distance ranges, 1.8–2.9, 1.8–3.5, and 1.8–5.0 Å, corresponding to strong, medium, and weak NOEs, respectively. For NOEs involving NH protons, a range of 1.8–5.9 Å was added, corresponding to ultraweak NOEs. Hydrogen bonding restraints (2 per hydrogen bond, where r_NH–O = 1.8–2.2 Å and r_N–O = 2.2–3.3 Å), and backbone dihedral angle restraints ( and angles) were generated from the standard secondary structure of the protein on the basis of the NOE patterns and the backbone secondary chemical shifts. Structures were calculated by using the program CNS (Brunger et al., 1998). The coordinates of Nyv1p-LD have been deposited in the Protein Data Bank under the accession code 2FZ0.

Yeast Strains and Methods
Yeast cells lacking VPS27 or PEP12 were grown at 25°C; all other yeast strains were grown at 30°C. Cells were propagated in yeast extract peptone dextrose medium (YEPD), synthetic dextrose media (SD), or synthetic galactose (SG) media lacking the appropriate amino acids. Yeast transformations were performed using lithium acetate by the method of Elble (1992). The yeast strains used in this study are listed in Table 2.

Plasmids Used in Protein Localization and Biochemical Studies
DNA fragments encoding Nyv1p-LD and nyv1p-LD^Y31Q;I34Q (amino acids 1–149) were amplified by PCR and cloned into pGEX2T as BamHI–EcoRI fragments to generate glutathione S-transferase (GST) fusion proteins pGST-Nyv1-LD and pGST-nyv1-LD ^YI/QQ, respectively. Plasmids encoding green fluorescent protein (GFP)-Nyv1p and GFP-Yck3p were based on the yeast/bacterial shuttle vectors pRS413 (CEN6, HIS3) or pRS416 (CEN6, URA3). The PCR-generated Yck3p- and Nyv1p-encoding derivatives were cloned as EcoRI–BamHI fragments behind sequences expressing the GFP mut2 variant (Cormack et al., 1996) and the triose phosphate isomerase promoter to generate the plasmids pGFP-Nyv1p, pGFP-nyv1p^YI/QQ (Y31Q, L34Q), pGFP-nyv1p^LI/QQ (L73Q, I74Q), and pGFP-Yck3p, respectively. The plasmid expressing hemagglutinin antigen (HA)-tagged APL6 under control of the GAL1 promoter [pGAL1-APL6-(His)₆-HA-protein A, in the plasmid BG1805] was purchased from Open Biosystems (Huntsville, AL). The plasmid expressing GFP-Pho8p, under the control of the PHO8 promoter, was a gift from Rob Piper (Department of Physiology, University of Iowa, Iowa City, IA). The plasmid expressing GNS (Reggiori et al., 2000) was a gift from Hugh Pelham (Medical Research Council-Laboratory of Molecular Biology, Cambridge, United Kingdom). The plasmid used as template for site-directed mutagenesis of the mu chain of AP3 (pRS416-APM3) was purchased from Euroscarf (Institute of Microbiology, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany) (pYCG_YBR288c). Plasmids expressing membrane-anchored GFP-longin domains were generated as follows. DNA fragments encoding amino acids 1–162 of Nyv1p, amino acids 1–126 of Sec22p, and amino acids 1–134 of Ykt6p were amplified by the PCR and cloned as EcoRI–HindIII fragments. The DNA encoding the Snc1p transmembrane spanning sequence (amino acids 93–117, together with a stop codon immediately after residue 117) was amplified by the PCR and cloned as a HindIII–BamHI fragment. The recombinant EcoRI–BamHI fragments were then used to replace the corresponding region of pGFP-Nyv1p. Amino acid substitutions in Nyv1p and Apm3p were generated using plasmids encoding the wild-type genes and the Kunkel method of site-directed mutagenesis (Kunkel, 1985).

Live Cell Imaging
Yeast cells were grown to mid-log phase, harvested by centrifugation, and resuspended in water. Aliquots of yeast cell suspensions (0.5–1.0 µl) were placed onto slides and examined immediately. Cells were visualized and photographed through a Zeiss Plan-Neofluar 100x/1.30A oil objective lens by using a Zeiss Axioskop microscope (Carl Zeiss, Jena, Germany) equipped with a SPOT-RT KE monochrome charge-coupled device camera (Diagnostic Instruments, Sterling Heights, MI). Digital images were processed with Photoshop (Adobe Systems, Mountain View, CA). The staining of yeast cells with N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM4–64) (Invitrogen, Carlsbad, CA) was carried out as described by Vida and Emr (1995).

In Vivo Protein Copurification
BY4741 cells (Table 2) transformed with pGFP-Nyv1p and SARY1401 cells (expressing Apm3-TAP; Table 2) transformed with pGFP-Nyv1p, pGFP-nyv1p^YI/QQ, or pRS416 were grown with constant shaking at 30°C in SD media lacking leucine (SD-Leu) until an OD₆₆₀ of 0.8 was reached. Cells were collected by centrifugation at 5000 x g for 20 min at 4°C, resuspended in NP-40 buffer [15 mM Na₂HPO₄, 10 mM NaH₂PO₄·H₂O, 150 mM NaCl, 1% (vol/vol) NP-40] containing 1% (vol/vol) Triton X-100 and a cocktail of protease inhibitors (EDTA-free Complete and Pefabloc; Roche Diagnostics, Mannheim, Germany), and lysed using a Mini-Bead Beater-8 (BioSpec Products, Bartlesville, OK). Unlysed cells and cellular debris were removed by centrifugation at 16,000 x g for 20 min at 4°C. IgG-beads (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) were added to yeast cell extracts, and the mixture was incubated, with constant mixing, at 4°C for 2 h. After incubation, IgG-beads were collected by centrifugation at 8000 x g for 1 min and washed four times with 10 bead volumes of NP-40 buffer containing 1% Triton X-100. After washing, IgG-beads were collected by centrifugation and resuspended in SDS-PAGE sample buffer. Proteins bound to the IgG-beads were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunostained with an anti-GFP antibody (Clontech, Mountain View, CA).

Purification of Proteins for In Vitro Mixing Studies
Plasmids encoding GST-Nyv1-LD, GST-nyv1-LD^(Y31Q,I34Q), or GST (pGEX2T) were transformed into E. coli BL21 cells. Protein expression was induced with 0.2 mM IPTG for 2 h at 30°C after which cells were collected by centrifugation at 5000 x g for 20 min at 4°C. Bacterial cell pellets were resuspended in NP-40 buffer containing 0.5% Triton X-100 and a protease inhibitor cocktail and lysed by sonication. Bacterial cell lysates were cleared of unbroken cells and cellular debris by centrifugation at 4°C.

Equal volumes of bacterial cell lysates (corresponding to 10 ml of bacterial culture) were incubated with 20 µl of glutathione (GSH)-Sepharose beads (GE Healthcare) for 2 h at 4°C. After incubation, beads were washed three times with NP-40 buffer (containing 0.5% Triton X-100) and finally resuspended in NP-40 buffer before use in in vitro pull-down assays (see below).

SARY1618 cells (Table 2) were grown in SD-Ura medium at 30°C to an OD₆₆₀ of 0.8. Yeast cells were collected by centrifugation, washed twice with distilled water to remove the glucose-containing medium, and resuspended in SG-Ura (2% galactose) to an OD₆₆₀ of 0.1. Cells were grown in SG-Ura for 12–14 h; collected by centrifugation; resuspended in 200 mM Tris-HCl, pH 8.0, 20 mM EDTA, and 1% (vol/vol) -mercaptoethanol; and incubated at 30°C for 15 min. After incubation, yeast cells were collected by centrifugation and resuspended in minimal medium containing 1 M sorbitol and 0.15 mg/ml Zymolyase 100T (Seikagaku, Tokyo, Japan) and incubated for 40 min at 30°C. After treatment with Zymolyase 100T, cells were washed twice with minimal medium containing 1 M sorbitol and resuspended in NP-40 buffer containing 0.5% Triton X-100. Cells were lysed in the presence of a protease inhibitor cocktail (EDTA-free Complete and Pefabloc) with 20 strokes of a Dounce homogenizer. Cell lysates were cleared by centrifugation at 16,000 x g for 20 min at 4°C.

In vitro mixing studies were performed as follows. GSH-beads containing GST-Nyv1-LD, GST-nyv1-LD^(Y31Q;I34Q), or GST were mixed with 100 OD₆₆₀ equivalents of whole cell extracts from SARY1618 cells, prepared as described above. The mixtures were incubated with constant mixing for 2 h at 4°C. After incubation, the beads were washed four times with NP-40 buffer [containing 0.5% (vol/vol) Triton X-100] for total of 0.5 h. After washing, beads were collected by centrifugation, boiled in SDS sample buffer, and processed for immunostaining with an anti-HA (Roche Diagnostics) antibody.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Nyv1p Is a Longin Domain-containing SNARE Protein
The structures of the N-terminal domains have been determined for Ykt6p (Tochio et al., 2001) and for the mammalian orthologue of Sec22p (Sec22b; Gonzalez et al., 2001). The structures of the N-terminal domains of Sec22b and Ykt6p are similar and bear overall fold similarity to that of the actin-binding protein profilin. To establish whether the third member of the longer R-SNARE family in yeast, Nyv1p, also contained a so-called longin domain, we determined the high-resolution solution structure of N-terminal domain of Nyv1p (residues 1–149) by NMR spectroscopy (Table 1).

View larger version (71K): [in this window] [in a new window]   Figure 1. Solution structure of the NH2-terminal longin domain of Nyv1p (Nyv1p-LD). (A) Stereo view of 20 superimposed NMR-derived structures of the Nyv1p-LD. The residues 4 N142 are shown in the drawing. The structures are superimposed against the averaged structure using residues 5 N26, 35 N60, and 65 N141. (B) Ribbon diagram representation of the Nyv1p-LD structure. (C) Close-up view of the hydrophobic surface of the Nyv1p-LD. The amino acid residues forming the hydrophobic surface are shown in the explicit atomic model. Molecular models in this and subsequent figures were generated using MOLMOL (Koradi et al., 1996), MOLSCRIPT (Kraulis, 1991), Raster3D (Merritt and Murphy, 1994), and DINO (www.dino3d.org).

View larger version (60K): [in this window] [in a new window]   Figure 2. Structural features of SNARE longin domains. (A) Ribbon diagram showing the longin domain structures from Nyv1p (Nyv1p-LD), Ykt6p (Ykt6p-LD), and Sec22b (Sec22b-LD). (B) The longin domain of Nyv1p contains a large surface exposed hydrophobic surface. In the surface representations of the longin domains from Nyv1p (Nyv1p-LD), Ykt6p (Ykt6p-LD), and Sec22b (Sec22b-LD), K and R are shown in blue; D and E are in red; F, V, L, I, A, M, W, and P are in yellow; and the rest of residues are shown in white. The orientation of the Nyv1p-LD is the same as that shown in Figure 1B and is similar to those of Ykt6p-LD and Sec22b-LD.

Nyv1p Does Not Adopt a Folded-back Conformation
The hydrophobic surface on the Ykt6p-LD has been suggested to play an autoinhibitory role by binding to the SNARE-motif of Ykt6p in cis (Tochio et al., 2001) and may also be important for chaperoning the lipid-modified C terminus of the protein (Hasegawa et al., 2004). In contrast, the longin domain of Sec22b does not contain such a hydrophobic surface, and the SNARE-motif of Sec22b does not bind to the longin domain but rather adopts an open conformation (Gonzalez et al., 2001). Given the presence of the large solvent-exposed hydrophobic surface, we reasoned that like Ykt6p, the longin domain of Nyv1p might also be able to bind to its SNARE-motif, thereby allowing Nyv1p to adopt an autoinhibited conformation. To test this hypothesis, we compared the ¹H, ¹⁵N-heteronuclear single-quantum coherence (HSQC) spectrum of the Nyv1p-LD to that of the full-length soluble form of Nyv1p, i.e., lacking its membrane spanning region (designated Nyv1pTMD). A subset of peaks from the Nyv1pTMD spectra overlaps well with the entire spectrum of Nyv1p-LD (Figure 3A). In particular, the amino acid residues that form the hydrophobic surface of Nyv1p-LD did not experience any detectable core domain-induced chemical shift changes, indicating that the SNARE-motif of Nyv1pTMD does not interact with its longin domain (Figure 3, A and B). The extra peaks in the Nyv1pTMD ¹H, ¹⁵N-HSQC spectra are located in a region indicative of a random coil structure that presumably corresponds to amino acid residues of the Nyv1p SNARE-motif. In Figure 3B, the small chemical shift changes observed in the C helix are a result of the covalent attachment of the SNARE core domain to the C helix. The even smaller chemical shift changes to the residues in the A/B-loop are due to the proximity of this region with the SNARE core domain. Because one would expect to observe much greater chemical shift changes (0.5 ppm or even higher; Wuthrich, 2000) if the C helix and A/B-loop were in direct contact with the SNARE core domain, we concluded that Nyv1pTMD adopts an open conformation in solution.

View larger version (31K): [in this window] [in a new window]   Figure 3. Nyv1pTMD does not adopt a folded-back conformation in solution. (A) Overlay of 1H, 15N-HSQC spectra of Nyv1p-LD (red), and Nyv1pTMD (black). (B) Summary of 1H and 15N combined chemical shift changes in the NH2-domain of Nyv1pTMD induced by the SNARE-motif. The combined 1H and 15N chemical shift changes are defined as ppm = [(HN)2 + (N x N)2], where HN and N represent chemical shift differences of amide proton and nitrogen chemical shifts of Nyv1p-LD with and without the SNARE-motif. The scaling factor (N) used to normalize the 1H and 15N chemical shifts is 0.17.

To establish whether Nyv1p, Ykt6p, or Sec22p contained localization determinants in their longin domains, we created fusion proteins in which the longin domains of these proteins were fused to GFP at their N termini and to the transmembrane domain of the R-SNARE Snc1p at their C termini (designated Nyv1p^LD, Sec22p^LD, and Ykt6p^LD in Figure 4A). We reasoned that those membrane-anchored longin domains that lacked dominant sorting signals would be localized to the cell surface by virtue of the Snc1p transmembrane spanning sequence (Reggiori et al., 2000). Plasmids encoding GFP-tagged, full-length SNAREs or encoding membrane anchored longin domains from either Nyv1p, Sec22p, or Ykt6p were transformed into yeast cells, and the intracellular location of the fusion proteins was visualized by microscopy (Figure 4B). As expected, GFP-Nyv1p localized to the limiting membrane of the vacuole, whereas GFP-Sec22p was localized to the ER. GFP-Ykt6p was found throughout the cell, and the intensity of cytoplasmic staining effectively masked any evidence of membrane localization (Figure 4B, left). Like the full-length protein, GFP-Nyv1-LD was also localized to the limiting membrane of the vacuole (Figure 4B, right). In contrast, the membrane-anchored longin domains of Sec22p and Ykt6p localized to the cell surface as well as to the lumen of the vacuole (Figure 4B, right). The GFP-Sec22-LD and GFP-Ykt6-LD protein found in the lumen of the vacuole likely corresponds to misfolded protein targeted for degradation, because traffic to this location could be blocked in cells that lacked the prevacuolar endosomal syntaxin Pep12p (Figure 4C).

View larger version (87K): [in this window] [in a new window]   Figure 4. The longin domain is sufficient to direct the sorting of Nyv1p to the limiting membrane of the vacuole. (A) Scheme used for the construction of GFP fusions to the longin domains of Nyv1p, Sec22p, and Ykt6p. (B) The localization of GFP-Nyv1p, GFP-Sec22p and GFP-Ykt6p and their membrane-anchored longin domains (Nyv1pLD, Sec22pLD, and Ykt6pLD) in wild-type cells. (C) Ykt6pLD and Sec22pLD localize to the plasma membrane in pep12 cells. Yeast cells were transformed with plasmids expressing the various GFP-fusion proteins and processed for microscopy as described in Materials and Methods

View larger version (53K): [in this window] [in a new window]   Figure 5. Sorting of GFP-Nyv1p and GFP-Nyv1-LD to the limiting membrane of the vacuole is mediated by AP3. (A) Schematic representation of trafficking routes from the Golgi to the vacuole in yeast. Trafficking routes are denoted by the use of arrows. Yeast gene deletion mutants (used in this study) defective in a particular trafficking route are indicated below a given arrow. Thus, cells lacking PEP12 (pep12) are defective in transport from the Golgi to the prevacuolar endosome designated CPY. Cells lacking APL5 (apl5) are defective in transport from the Golgi to the vacuole designated AP3, and cells lacking VPS27 (vps27) accumulate proteins destined for the vacuole via endosomes, in an aberrant structure, termed the class E compartment. Yeast cells lacking APL5, VPS27, or PEP12 were used to assess trafficking routes taken by GFP-Nyv1p, GFP-Nyv1LD, and GNS (and their corresponding amino acid substitution mutants); see text for details. (B) The localization of GFP-Nyv1p (Nyv1p) and GFP-Nyv1-LD (Nyv1pLD) in wild-type cells and in the indicated yeast single gene deletion strains (see Table 2 and text for details). To visualize vacuoles, cells were stained with FM4-64 (see Materials and Methods). Arrowheads in the FM4-64 panels (vps27) indicate the location of the class E compartment. Note the absence of the GFP fusion proteins from the class E compartment in the corresponding vps27 GFP panels.

apl5

Thus, unlike the Nyv1p-LD, when the longin domains of Sec22p and Ykt6p are tethered to membranes via the Snc1p transmembrane domain, they do not seem to convey any dominant protein sorting signals. However, we cannot exclude the possibility that the longin domain of Sec22p is required for COPII-mediated export from the ER (Miller et al., 2003; Mossessova et al., 2003; Liu et al., 2004).

The Longin Domain of Nyv1p Contains a YXX-Motif That Is Required for Its AP3-dependent Sorting to the Limiting Membrane of the Vacuole
Having established that the longin domain of Nyv1p contained an AP3-dependent sorting signal, we wanted to identify the location and nature of the motif. The longin domain of Nyv1p contains three potential YXX-motifs: Y⁸VEV¹¹, Y³¹GTI³⁴, and Y⁹²VCF⁹⁵; two of these consensus amino acid sequences map to the A (Y⁸VEV¹¹) and E (Y⁹²VCF⁹⁵) regions of the longin fold, and both are buried in the core of the protein, whereas the third sequence, Y³¹GTI³⁴, is located in a surface-exposed and highly dynamic region between B and A (Figure 6, A and B). A structural-based amino acid sequence alignment of the longin domains of Nyv1p, Ykt6p, and Sec22b revealed that the Y³¹GTI³⁴ YXX-like motif is uniquely placed in the longin domain of Nyv1p (Figure 6C). To determine whether the Y³¹GTI³⁴ sequence functioned as an AP3-dependent sorting signal in Nyv1p, a double amino acid substitution of Y31 to Q and I34 to Q (nyv1p^YI-QQ) was generated by site-directed mutagenesis, and the localization pattern of the mutant GFP-tagged protein was visualized in yeast by microscopy (Figure 7A).

View larger version (48K): [in this window] [in a new window]   Figure 6. A YXX-motif is located in a unique, surface exposed and highly dynamic region of the Nyv1p longin domain. (A) Superimposed NMR-derived structures of Nyv1p-LD showing the location of the surface exposed and highly dynamic loop (boxed) containing the Y31GTI34 YXX-motif. (B) Surface representation of the Nyv1p-LD in which the locations of Y31, G32, T33, and I34 are prominently represented. The model shown represents a 90° clockwise rotation of the image shown in A. Lys and Arg are shown in blue; Asp and Glu are in red; Phe, Val, Leu, Ile, Ala, Met, Trp, and Pro are in yellow; and the rest of residues are shown in white. (C) A structure-based alignment of the longin domains of Nyv1p (amino acids 1–149), Ykt6p (amino acids 1–140), and Sec22b (amino acids 1–127). For reference, the secondary structure of the Nyv1p-LD is shown above the alignment. The amino acid residues of the Y31GTI34 YXX-motif in the Nyv1p-LD (between B and A) are shaded in purple. Amino acids shaded in yellow denote hydrophobic residues that are conserved between Nyv1p-LD, Ykt6p-LD, and Sec22b-LD, whereas amino acids shaded in red denote conserved charged residues.

View larger version (109K): [in this window] [in a new window]   Figure 7. Amino acid substitutions to the Y31GTI34 YXX-like motif in Nyv1p and Nyv1p-LD disrupt their AP3-dependent sorting. (A) The localization and sorting requirements of GFP-nyv1p Y31Q;I34Q (designated nyv1YI-QQ), in wild type and in the indicated yeast single gene deletion strains. The arrowheads in the vps27 panel denote the position of the class E compartment, which is visible in both the FM4-64 and GFP images. (B) The localization and sorting requirements of GFP-nyv1-LD Y31Q;I34Q (designated LDYI-QQ), in wild type and in the indicated yeast single gene deletion strains. The arrowheads in the vps27 panel indicate the position of the class E compartment, which is visible in both the FM4-64 and GFP images.

We also examined the effect of the Y31 to Q and I34 to Q double amino acid substitution on the sorting of GFP-Nyv1-LD (Figure 7B). As expected, GFP-nyv1-LD^YI-QQ was localized to the cell surface, indicating that Y31 and I34 are required to direct GFP-Nyv1-LD to the vacuole. However, in addition to cell surface localization, GFP-nyv1-LD^YI-QQ was also found on the limiting membrane of the vacuole as well as in the lumen of the vacuole. Consistent with this observation, GFP-nyv1-LD^YI-QQ accumulated in the class E compartment of vps27 cells (Figure 7B). Evidently, some GFP-nyv1-LD^YI-QQ is targeted for degradation in the vacuole via the CPY pathway (pep12; Figure 7B), indicating that this protein is either misfolded and/or recognized as being aberrant. Regardless, some GFP-nyv1-LD^YI-QQ is clearly mislocalized to the cell surface, supporting the notion that Y³¹GTI³⁴ functions as an AP3-dependent sorting signal in the longin domain of Nyv1p. The vacuolar localization observed for GFP-nyv1-LD^YI-QQ in pep12 cells may reflect the use of additional AP3-dependent sorting signals (see below) or alternatively, represent protein transported to the vacuole by default via the AP3 pathway (Bruinsma et al., 2004). In contrast, the relatively faint vacuolar localization observed for GFP-nyv1-LD^YI-QQ in apl5 cells most likely corresponds to protein redirected to the vacuole by default via the CPY pathway (Figures 5A and 7B).

To address the potential contribution of the transmembrane domain of nyv1p^YI-QQ to the localization of protein to the limiting membrane of the vacuole in wild-type and pep12 cells (Figure 7A) as well as to better visualize the extent of mislocalization of the Nyv1p site-directed mutant protein, we used a GFP-tagged version of Nyv1p in which the Nyv1p transmembrane spanning sequence was replaced with that of Snc1p (GFP-Nyv1-Snc1^TMD) termed GNS (Reggiori et al., 2000). Yeast mutants that effect the sorting of GNS and as well as mutations in GNS that effect the localization of the protein will result in their redirection from the vacuole to the cell surface.

As expected, the localization of GNS was indistinguishable from that of GFP-Nyv1p in wild-type cells (compare Figure 4B with Figure 8A). In wild-type cells, GNS was found on the limiting membrane of the vacuole, and its sorting to this location required a functional AP3 complex; as when expressed in cells lacking the APL5 gene (which encodes the subunit of yeast AP3), GNS was largely mislocalized to the plasma membrane (Figure 8A). As anticipated, the sorting of GNS to the limiting membrane of the vacuole did not proceed via the CPY pathway (Figure 5A), because the protein failed to accumulate in the class E compartment of vps27 cells (Figure 8A) and its localization to the limiting membrane of the vacuole was unaffected in cells lacking PEP12 (pep12; Figure 8A). In contrast to the double amino acid substitution mutant of Nyv1p (GFP-nyv1^YI-QQ), which was found primarily in the lumen of the vacuole (Figure 7A), GNS^YI-QQ was mislocalized to the plasma membrane in wild-type cells (Figure 8B). The extent of mislocalization of GNS^YI-QQ in wild-type cells was essentially indistinguishable from that of GNS and GNS^YI-QQ in apl5 cells (compare Figure 8, A and B), suggesting that the Y³¹GTI³⁴ motif is the predominant AP3-dependent sorting signal in the protein. In contrast to GFP-nyv1p^YI-QQ (Figure 7A), GNS^YI-QQ did not localize to the limiting membrane of the vacuole in pep12 cells (Figure 8B). We therefore concluded that either the transmembrane domain of Nyv1p also contributed modestly to the AP3-dependent sorting of the protein in wild-type cells, or alternatively, that GFP-nyv1p^YI-QQ but not GNS^YI-QQ was sorted by default via the AP3 pathway in pep12 cells (Bruinsma et al., 2004).

View larger version (79K): [in this window] [in a new window]   Figure 8. Localization and sorting requirements of GFP-Nyv1-Snc1TMD (GNS) and its amino acid substitution mutants GFP-Nyv1Y31Q;I34Q-Snc1TMD (GNSYI-QQ) and GFP-Nyv1L71Q;I74Q-Snc1TMD (GNSLI-QQ). (A) The localization pattern of GNS in wild-type, apl5, vps27, and pep12 cells. The arrowheads in the vps27 panel indicate the position of the class E compartment, which is visible in the FM4-64 panel but not in the GFP panel. (B) The localization pattern of GNS Y31Q;I34Q (designated GNSYI-QQ) in wild type, apl5, vps27, and pep12 cells. The arrowheads in the vps27 panel indicate the location of the class E compartment, which is visible in the FM4-64 panel and into which GNSYI-QQ also accumulates, albeit modestly (arrowhead in the corresponding GFP panel). (C) The folding of the Nyv1p-LD is not effected by the introduction of the Y31Q;I34Q amino acid substitutions. The structure perturbations caused by the Y31Q;I34Q substitutions are predominantly restricted to the loop containing the YXX-like motif, indicating that the structural integrity of the longin fold is largely unaffected in GNSYI-QQ, see the text for details. (D) Amino acid substitutions that disrupt the surface exposed hydrophobic patch of the Nyv1p-LD (L71;I74Q) have no effect on the localization of GNSLI-QQ.

Because the introduction of amino acid substitutions can sometimes result in changes to the folding of a protein, we wanted to eliminate mutation-induced folding alterations as a possible explanation for the degree of mislocalization observed for GNS^YI-QQ (Figure 8B). To address this possibility, we investigated conformation changes to the Nyv1p LD that resulted from the Y31Q, I34Q mutations, by using an NMR chemical shift perturbation approach (Figure 8C). From these data, it is clear that with the exception of the B/A-loop, the Y31Q, I34Q amino acid substitutions did not alter the conformation of the longin fold (Figure 8C).

Like Ykt6p-LD, Nyv1p-LD also contains a prominent hydrophobic surface (yellow in Figure 2B), and we next addressed whether this surface in the Nyv1p-LD might also be required for interaction of the longin domain with the AP3 complex. We reasoned that amino acid substitutions that disrupted the hydrophobic surface would be expected to disrupt interactions between Nyv1p/GNS and AP3, resulting in mislocalization of Nyv1p/GNS. Using site-directed mutagenesis, we introduced amino acid substitutions at L73 (L73Q) and I74 (I74Q) to generate GNS^LI-QQ. Unlike GNS^YI-QQ, GNS^LI-QQ was localized to the limiting membrane of vacuole (Figure 8D), and we therefore concluded that L73 and I74 (and therefore the solvent-exposed hydrophobic surface) were not critical for interactions between Nyv1p/GNS and the AP3 sorting machinery.

An alternate explanation for the residual localization of GFP-Nyv1^YI-QQ and GNS^YI-QQ to limiting membrane of the vacuole in wild-type cells (Figures 7A and 8B) is the presence of one or more additional (albeit weaker) AP3-dependent sorting signals in these proteins. In addition to the Y³¹GTI³⁴ motif, the longin domain of Nyv1p also contains two additional YXX consensus sequences: Y⁸VEV¹¹ and Y⁹²VCF⁹⁵. As discussed above, the structure of the Nyv1-LD has revealed that both of these motifs are buried in the protein and thus would presumably only function as AP3-dependent sorting signals if the longin domain underwent a substantial conformational change. Nonetheless, to address the possible roles of the Y⁸VEV¹¹ and Y⁹²VCF⁹⁵ motifs as contributors to the AP3-dependent sorting of GNS and Nyv1-LD, double amino acid substitutions were introduced by site-directed mutagenesis, creating nyv1-LD^YV-QQ (Y8Q;V11Q), nyv1-LD^YF-QQ (Y92Q;F95Q), GNS^YV-QQ (Y8Q;V11Q), and GNS^YF-QQ (Y92Q;F95Q). The localization patterns of the mutant GFP-tagged proteins were then visualized in wild-type cells as well as in various yeast mutants (Figure 5A) by microscopy (Figure 9, A and B). The localization patterns observed for both the GFP-nyv1-LD (LD; Figure 9A) and GNS amino acid substitution mutants (GNS; Figure 9B) were essentially indistinguishable from the localization patterns observed previously for GFP-Nyv1-LD (Figure 5B) and GNS (Figure 8A). Although the degree of mislocalization of LD^YV-QQ in apl5 is not as profound as LD^YF-QQ in apl5 cells, the localization pattern of the corresponding GNS mutant (GNS^YV-QQ, Figure 9B) is entirely consistent with the view that Y⁸VEV¹¹ does not function as an AP3-dependent sorting signal. Note that, as for GNS and GFP-Nyv1-LD in apl5 cells (Figures 5B and 8A), some the corresponding YV-QQ and YF-QQ mutant proteins were redirected to the vacuolar membrane via the CPY pathway, presumably by default (Stepp et al., 1997; Darsow et al., 1998; Vowels and Payne, 1998; Sun et al., 2004), because no class E compartment staining was observed for any of these mutants (vps27; Figure 9, A and B).

View larger version (127K): [in this window] [in a new window]   Figure 9. Amino acid substitutions targeting the additional YXX-like motifs in Nyv1p (Y8VEV11 and Y92VCF95) do not disrupt AP3-dependent sorting of the mutant proteins. (A) The localization and sorting requirements of the Y8Q;V11Q (LDYV-QQ) and Y92Q;F95Q (LDYF-QQ) longin domain amino acid substitution mutants. (B) The localization and sorting requirements of the Y8Q;V11Q (GNSYV-QQ) and Y92Q;F95Q (GNSYF-QQ) GFP-Nyv1-Snc1TMD amino acid substitution mutants. For both A and B, the arrowheads in the vps27 panels indicate the location of the class E compartment. Note the absence of GFP localization to the class E compartment in all vps27 panels.

Amino Acid Substitutions in the Longin Domain of Nyv1p That Disrupt the Sorting of GFP-Nyv1p In Vivo Reduce Binding to the AP3 Complex
Having established that the longin domain of Nyv1p was sufficient to mediate AP3-dependent sorting via the Y³¹GTI³⁴ YXX-like motif, we next wanted to determine whether Nyv1p could bind to the AP3 sorting machinery in vivo. To accomplish this, we expressed GFP-Nyv1p and GFP-nyvlp^YI-QQ in yeast cells (SARY1401; Table 2) in which the chromosomal copy of the mu chain of AP3 (Apm3p) had been modified through the addition of a tandem affinity purification tag (termed Apm3-TAP; Gavin et al., 2002). The localization pattern of GFP-Nyv1p in yeast cells expressing Apm3-TAP as their sole source of Apm3p was indistinguishable from that in wild-type cells (our unpublished data). Whole cell extracts were prepared from SARY1401 cells, and proteins that copurified with Apm3-TAP were isolated on IgG beads by virtue of the protein-A portion of the TAP tag to bind to immunoglobulin-coupled beads. As a control for nonspecific binding of GFP and/or GFP-Nyv1p proteins to IgG-beads, these experiments were carried out in parallel using whole cell extracts prepared from wild-type cells (BY4741; Table 2) expressing GFP-Nyv1p (Apm3; Figure 10, A and B). Proteins bound to IgG-beads were resolved by SDS-PAGE and immunostained with anti-GFP antibodies. Although both GFP-Nyv1p and GFP-nyv1p ^YI-QQ could be copurified with Apm3-TAP, the relative amount of GFP-nyv1p^YI-QQ bound to the AP3 complex was by comparison an average of 13-fold less (7.42 ± 7.82%) than the amount of GFP-Nyv1p that copurified with Apm3-TAP (from 5 independent experiments). The results from one of these experiments are shown in Figure 10A. The data for the five experiments are summarized graphically in Figure 10B. For comparison purposes, the binding of GFP-Nyv1p to Apm3-TAP was arbitrarily set to 100%; hence, there are no error bars for this data set in Figure 10B (i.e., WT in Apm3-TAP). Because the binding of GFP-Nyv1p to IgG-beads was undetectable (WT in Apm3; Figure 10, A and B), we concluded that neither Nyv1p nor GFP bound nonspecifically to IgG-beads under the conditions used here.

View larger version (47K): [in this window] [in a new window]   Figure 10. Amino acid substitutions that disrupt the sorting of Nyv1p in vivo reduce binding to the AP3 adaptin complex. (A) GFP-nyv1YI-QQ is defective in binding to the AP3 complex in vivo. Whole cell extracts were prepared from either BY4741 (Apm3, as a negative control) or SARY1401 cells (Amp3-TAP) transformed with GFP-Nyv1p (Apm3/WT) in the case of BY4741 cells and either GFP-Nyv1p (Apm3-TAP/WT) or GFP-nyv1p Y31Q;I34Q (Apm3-TAP/YI/QQ) in the case of SARY1401 cells. After incubation, proteins bound to IgG-beads were resolved on SDS-PAGE gels, transferred to nitrocellulose membranes, and immunostained with an anti-GFP antibody (as described in Materials and Methods). Note that Apm3-TAP binds to the secondary antibody used in these experiments (anti-IgG) and serves as a gel loading control. (B) Graphic representation of the copurification data from five independent experiments quantified by scanning densitometry. Binding of wild-type GFP-Nyv1p to AP3 was arbitrarily set as 100% and other data points are plotted relative to wild-type protein binding. The data (for n = 5) were WT (GFP-Nyv1p, 100%) and YI/QQ (GFP-nyv1pY31Q,I34Q, 7.42 ± 7.82%). Binding of GFP-Nyv1p to IgG-beads was undetectable (Apm3/WT; A and B). See text for details. (C) The longin domain of Nyv1p binds to AP3 in vitro. Equivalent amounts (10 µg) of bacterially expressed, purified recombinant GST, GST-Nyv1p-LD (LD), and GST-nyv1p(Y31Q, I34Q)-LD (YI/QQ) were immobilized on GSH-beads and mixed with equal amounts of whole cell extract prepared from wild-type cells expressing a galactose-inducible HA-tagged form of Apl6p from a plasmid (SARY1618 cells; see Table 2). Proteins bound to GSH-beads were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunostained with an anti-HA antibody. An aliquot of whole cell extract from SARY1618 cells (WCE) was also included as a gel mobility reference for HA-tagged Apl6p. Bottom, Coomassie brilliant blue-stained SDS-PAGE gel of identical amounts of recombinant proteins bound to GSH-beads that were used in the pull-down assays shown above. (D) Graphic representation of the copurification data from three independent experiments, quantified by scanning densitometry. The binding of the wild-type longin domain of Nyv1p (LD) to AP3 was arbitrarily set as 100%, and other data points are plotted relative to wild-type protein binding. The data were LD (100%), GST (4.97 ± 7.52%) and YI/QQ (42.98 ± 15.29%).

Although the reduction in binding observed for the Nyv1p YI/QQ mutant in our in vivo and in vitro experiments was modest, our data from the in vivo coprecipitation experiments (Figure 10A) are in line with another study that examined interactions between mammalian AP3 and one of its YXX-containing ligands (Nishimura et al., 2002). We therefore consider our biochemical data to be consistent with our in vivo protein sorting data. Together, these experiments support the view that amino acid substitutions to the Y³¹GTI³⁴ motif that disrupted sorting of Nyv1p in vivo were most likely the result of a reduced binding affinity between the Nyv1p longin domain AP3.

Amino Acid Substitutions in the mu Chain of Yeast AP3 Preferentially Effect Sorting of YXX-containing Cargoes
Because GFP-Nyv1p could be copurified with AP3, we next wanted to determine which subunit(s) of AP3 was responsible for recognition of the Y³¹GTI³⁴ YXX-like motif in the longin domain of Nyv1p. To accomplish this, we took advantage of what had been learned about the molecular basis of the recognition and sorting of YXX signal-containing cargo by the mu chain of the mammalian AP2 complex (Owen and Evans, 1998; Owen et al., 2004; Honing et al., 2005), because relatively little is known about the molecular basis of recognition of YXX-containing cargo proteins by the AP3 adaptor complex. Using a structure-based amino acid sequence alignment of the C-terminal domain of the mu chain of rat AP2 as a model for YXX ligand binding, amino acid substitutions were introduced into the mu chain subunit (Apm3p) of yeast AP3 (µ1–µ4, Figure 11A). The effects of these amino acid substitutions on the sorting of YXX- and acidic dileucine-containing yeast AP3 cargo proteins were assessed by microscopy. In these experiments, the AP3-dependent acidic dileucine-containing cargo protein GFP-Pho8p served as a control for assessing whether any of our mu chain amino acid substitutions resulted in loss-of-function of AP3 (as in rat AP2, binding of the acidic dileucine motif occurs at a different site from that of YXX-containing proteins; Honing et al., 2005). Mislocalization in individual µ-amino acid substitution mutants was assessed on the basis of how closely the GFP-fusion protein localization phenotype (Figure 11A) coincided with that observed in apm3 cells expressing the same GFP-tagged marker protein. For example, GNS and GFP-Yck3p were considered mislocalized if any cell surface staining was apparent, even if some protein was still localized to the vacuole (as is the case for both GNS and GFP-Yck3p in apm3 cells; Figure 11E; Sun et al., 2004). As discussed above, the residual localization of GNS to the limiting membrane likely represented protein that had been redirected, via the CPY pathway, to the vacuolar membrane in the absence of a functional AP3 pathway (Figures 5A and 8, A and B; Sun et al., 2004).

View larger version (96K): [in this window] [in a new window]   Figure 11. Effect of amino acid substitutions in the mu chain of yeast AP3 (Apm3p) on YXX-motif and acidic dileucine motif containing cargo proteins. (A) Top, an alignment of a portion of the C-terminal regions of the mu chains from yeast AP3 (Sc AP3µ), rat AP2 (Rat AP2µ), and rat AP3 (rat AP3µ). The amino acid residue numbering corresponds to that of the yeast AP3 mu chain (Apm3p). Bottom, summary of amino acid substitution mutations introduced into the mu chain of yeast AP3, the position and nature of amino acid substitutions are indicated with bold font (see text for details). (B) Effect of the Apm3p amino acid substitution mutants µ1 and µ2 on the sorting of GNS. (C) Effect of single amino acid substations in the Y31GTI34 YXX-like motif on the localization of GNS. (D) Percentage of mislocalization of YXX and acidic dileucine-containing cargo proteins in the µ3 and µ4 mu chain amino acid substitution mutants (see A and text for details). Yeast cells expressing the indicated APM3 mutant as their sole source of Apm3p were transformed with plasmids expressing GNS (YXX signal-containing cargo protein), GFP-Yck3p (YXX signal-containing cargo protein) or GFP-Pho8 (acidic dileucine signal-containing cargo protein). Mislocalization was assessed on the basis of how closely the localization phenotype of individual cells coincided with those of apm3 mutants expressing the same GFP-tagged marker protein (see text for details). GFP-Yck3p and GNS were considered to be mislocalized if cell surface localization was apparent. GFP-Pho8p was considered to be mislocalized if localization to the lumen of the vacuole was apparent. For wild-type cells, the data scored were GFP-Yck3p (mislocalized, n = 18; wild type, n = 151) GNS (mislocalized, n = 19; wild type, n = 194), GFP-Pho8p (mislocalized, n = 20; wild type n = 187). For the µ3 mutant the data collected were GFP-Yck3p (mislocalized, n = 148; wild type, n = 78), GNS (mislocalized, n = 51; wild type, n = 169), GFP-Pho8p (mislocalized, n = 30; wild type, n = 189). For the µ4 mutant, the data collected were GFP-Yck3p (mislocalized, n = 150; wild type, n = 0) GNS (mislocalized, n = 175; wild type, n = 56), GFP-Pho8p (mislocalized, n = 15; wild type, n = 108). (E) Mislocalization of YXX-motif containing cargo proteins in apm3 cells expressing the µ4 mutant (apm34). Note that GFP-Yck3p and GNS localizes to the cell surface (as well as the vacuole) in the majority of µ4 mutant cells, whereas GFP-Pho8p localization in the µ4 mutant cells is essentially indistinguishable from that in wild-type cells.

Our findings on the sorting of AP3-dependent YXX-containing cargo proteins in cells expressing the Apm3p D217 and Apm3p Y215, D217 substitution mutants were perplexing particularly as substitution of Y31 to Q was sufficient to mislocalize the majority of GNS^Y31Q (Figure 11C), highlighting the relative importance of the tyrosine residue in the Y³¹GTI³⁴ motif. Substitution of I34 with Q, in contrast, had significantly less impact on the localization of GNS^I34Q, were the majority of cells showed localization to the limiting membrane of the vacuole (Figure 11C). On the basis of these results, we concluded that D217 of Apm3p was not crucial for the binding of AP3-interacting YXX-motifs in vivo. To establish whether amino acid substitutions elsewhere in the presumptive YXX binding site of Apm3p would effect the localization of GNS in cells, we generated additional amino acid substitutions t