Fig. 1. Vesicles accumulate inside protease-deficient vacuoles. (A) EM sections of wild-type (WT) and Δpep4 yeast. Vacuoles appear as large white areas in these permanganate-fixed cells, which are empty in the wild-type cells but contain aggregates with vesicular profiles in the mutant (arrows). (B) Isolated vacuoles purified from a Δpep4 strain expressing a GFP-tagged mutant form of Pep12p show internal fluorescence. The inset shows an enlarged single confocal scan of a vacuole, revealing punctate structures within it.

Fig. 3. Delivery of proteins to the vacuole interior occurs via endosomes. GFP-tagged versions of the four proteins identified in internal vesicles were expressed in the indicated deletion mutant strains. The vps4 and pep12 cells were double labelled with the dye FM4-64 to reveal vacuoles and endosomal compartments. The diagram at the bottom indicates the position of the GFP tag (G) and the topology of the proteins in the membrane, as predicted by the TMHMM program (http://www.cbs.dtu.dk/services).

Fig. 5. The Sna family of proteins in yeast. (A) Alignment of the Sna3p sequence with the other three, with identities and similarities indicated by black and grey backgrounds. Thick bars over the sequences indicate the TMD. Dots indicate cytoplasmic lysines in Sna3p; the acidic di-leucine motif in Sna4p is underlined. (B) GFP-tagged Sna4p is on the vacuolar membrane in wild-type cells, but inside the vacuoles in Δapm3 cells. (C) GFP–Phm5p shows its normal intra-vacuolar pattern in a Δsna3 mutant.

Fig. 7. Ubiquitylation of Cps1p and Phm5p. (A) Δdoa4 cells expressing myc-tagged ubiquitin and either protein A-tagged Cps1p, or only endogenous Cps1p, were processed in parallel. Total extracts, and protein purified by binding to IgG–Sepharose followed by cleavage with TEV protease to release Cps1p from the protein A tag, were immunoblotted with anti-protein A (PA) or anti-myc. Note that protein A is absent from the purified material. Cps1p migrates as a doublet, due to variable glycosylation (Spormann et al., 1992). The faint bands above the protein A chimera are likely to be ubiquitylated forms, but these are under-represented in total cell extracts due to de-ubiquitylation during sample preparation. (B) As (A), but cells expressed protein A-tagged Phm5p. The lower protein A-containing band results from processing close to the transmembrane domain (see Figure 4). In addition, some more random degradation was detectable, which accounts for the smaller myc-tagged proteins in the purified fraction. In both panels, the asterisks indicate the approximate mobility expected for a mono-ubiquitylated protein.

Fig. 9. Ubiquitylation of Sna3p is not required for it to enter the vacuole. (A) Double labelling with YFP–Phm5p and GFP–Sna3p in wild-type (WT) and Δdoa4 cells. (B) GFP–Sna3p with both cytoplasmic lysines changed to arginines expressed in wild-type cells and in a Δsna3 mutant.

Fig. 2. Isolation of internal vesicles from vacuoles. (A) The fractionation procedure described in the text and Materials and methods was carried out on pep4 cells expressing GFP–PEP3D. Immunoblotting was used to follow the distribution of proteins in total cell extract (total), purified vacuoles, the P15 pellet, the S500 supernatant and the P500 pellet. The P500 and S500 samples were overloaded as indicated; the total extract was substantially underloaded since only a few percent of the vacuoles were recovered in the initial purification step. Intact GFP–PEP3D is under-represented in the total vacuole fraction due to proteolysis during sample preparation (data not shown). Markers are alkaline phosphatase (ALP, vacuolar membrane marker), carboxypeptidase Y (CPY, vacuole lumen), phosphoglycerate kinase (Pgk1p, cytosolic), dolicholphosphomannose synthase (Dpm1p, ER) and the syntaxin Sso2p (plasma membrane). (B) Coomassie Blue-stained gel of the S500 and P500 fractions from a scaled-up preparation. Molecular weight markers are indicated on the left (kDa). Proteins in the P500 sample were identified by tryptic digestion and mass spectrometry. These were often present in multiple bands, presumably due to proteolysis, and these are indicated by the linked lines. Asterisks mark bands containing the GFP–PEP3D marker, which also contaminated all three of the Lap4p bands. Numbered bands were identified as follows: 1, Fas1p (fatty acyl CoA synthase); 2, Ena2p (plasma membrane Na⁺-ATPase); 3, Pma1p (plasma membrane H⁺-ATPase); 4, a mixture of Tef2p (translation elongation factor) and Eno2p (enolase); 5, Adh1p (alcohol dehydrogenase); 6, Tdh3p; 7, Tdh2p (glyceraldehyde-3-P dehydrogenases). Phm5p was not identified from this gel, but the relevant part of a silver-stained gel from which it was obtained is shown on the right.

Fig. 4. Analysis of GFP chimeras. Western blots of total extracts from cells expressing the GFP-tagged versions of the indicated proteins were probed with anti-GFP antibodies. Triangles indicate full-length chimeras; asterisks mark a yeast protein that cross-reacts with the anti-GFP antibody. Bands at position 1 correspond to free GFP, those at position 2 contain GFP linked to the cytoplasmic tail and TMD of Cps1p or Phm5p. Molecular weight markers (kDa) are indicated on the right. The nature of the bands migrating more slowly than GFP–Sna3p is unknown; they may represent conformational isomers or protein complexes.

Fig. 6. Delivery to the vacuole interior is inhibited in Δdoa4 cells. GFP-tagged Cps1p, Phm5p and Hmx1p were expressed in Δdoa4 cells, which were also labelled with FM4-64. GFP fluorescence is more prominent on the vacuolar membrane than inside the vacuoles, in contrast to wild-type cells (see Figure 3). The brightest rings visible with GFP–Hmx1p correspond to the nuclear envelope.

Fig. 8. Sorting of Phm5p is blocked by mutation of Lys6, but restored by the addition of ubiquitin. GFP–Phm5p, the K6R mutant, and the mutant with ubiquitin added to the N-terminus of the GFP were expressed in both wild-type cells (WT) and the Δdoa4 mutant. The sequence of the Phm5p cytoplasmic tail is shown at the bottom.