Direct binding to Rsp5 mediates ubiquitin-independent sorting of Sna3 via the multivesicular body pathway

Abstract

The sorting of most integral membrane proteins into the lumenal vesicles of multivesicular bodies (MVBs) is dependent on the attachment of ubiquitin (Ub) to their cytosolic domains. However, Ub is not required for sorting of Sna3, an MVB vesicle cargo protein in yeast. We show that Sna3 circumvents Ub-mediated recognition by interacting directly with Rsp5, an E3 Ub ligase that catalyzes monoubiquitination of MVB vesicle cargoes. The PPAY motif in the C-terminal cytosolic domain of Sna3 binds the WW domains in Rsp5, and Sna3 is polyubiquitinated as a consequence of this association. However, Ub does not appear to be required for transport of Sna3 via the MVB pathway because its sorting occurs under conditions in which its ubiquitination is impaired. Consistent with Ub-independent function of the MVB pathway, we show by electron microscopy that the formation of MVB vesicles does not require Rsp5 E3 ligase activity. However, cells expressing a catalytically disabled form of Rsp5 have a greater frequency of smaller MVB vesicles compared with the relatively broad distribution of vesicles seen in MVBs of wild-type cells, suggesting that the formation of MVB vesicles is influenced by Rsp5-mediated ubiquitination.

Figure 1.

Sna3 is polyubiquitinated. (A) Schematic diagram of Sna3 indicating its orientation in the membrane and the locations of K₁₉, K₁₂₅, and the PPAY motif spanning amino acids 106-109. The N-terminal cytosolic domain of Sna3 consists of amino acids 1-21, and its C-terminal cytosolic domain consists of amino acids 64-133. (B) Western blot analysis of extracts prepared from wild-type cells (SEY6210), pep4Δ cells (TVY1), and pep4Δ doa4^C571S cells (MWY6) transformed with empty low-copy vector (pRS416) or with pRS416 encoding Sna3-GFP (pSNA3-GFP; Katzmann et al., 2004). Asterisks indicate ubiquitinated forms of Sna3-GFP and CPS. PGK was examined as a control to indicate loading of equivalent lysate amounts. The reduced intensity of CPS in pep4Δ doa4^C571S extracts compared with wild-type and pep4Δ extracts might be due to the fact that, in pep4Δ doa4^C571S cells, some of the total pool of CPS is monoubiquitinated and some is not. (C) Western blot analysis of extracts prepared from pep4Δ SNA3-GFP cells (MMY161) transformed either with empty high-copy vector (pRS202) or pRS202 encoding wild-type DOA4 (pGO289; Luhtala and Odorizzi, 2004). Asterisks indicate ubiquitinated forms of Sna3-GFP. Note that the low endogenous expression of Doa4 is difficult to detect. (D) Western blot analysis of extracts prepared from pep4Δ doa4^C571S SNA3-HA cells (MMY64) that had been transformed either with empty low-copy vector (pRS414) or pRS414 encoding Myc-Ub (pUB223; Berset et al., 2002) and incubated with 250 μM CuSO₄ to induce overexpression of Myc-Ub. Unmodified full-length Sna3-HA migrates at ∼25 kDa. Equivalent loading of each extract was confirmed by blotting for PGK (data not shown). In B–D, antibodies used for Western blotting are indicated on the left, and the migration of molecular weight standards (kDa) are indicated on the right. Blotting of the samples shown in B with anti-Ub antibodies confirmed that Sna3-HA was polyubiquitinated. (E) Fluorescence and DIC microscopy of wild-type cells (SEY6210) and doa4^C571S cells (GOY100) transformed with pSNA3-GFP or a high-copy vector encoding GFP-CPS (pGO47; Odorizzi et al., 1998). Bar, 2.5 μm.

Figure 2.

Rsp5 mediates polyubiquitination at either K₁₉ or K₁₂₅ of Sna3. (A) Western blot analysis of extracts prepared from pep4Δ doa4^C571S sna3Δ cells (MMY153) transformed with wild-type pSNA3-GFP or mutagenized pSNA3-GFP in which arginine was substituted for K₁₉ (pMM158), K₁₂₅ (pMM152), or both K₁₉ and K₁₂₅ (K₀; pMM159). (B) Western blot analysis of extracts prepared from pep4Δ doa4^C571S RSP5 TUL1 BSD2 cells (MMY64), pep4Δ doa4^C571S rsp5^G555D TUL1 BSD2 cells (MMY127), pep4Δ doa4^C571S rsp5^G555D tul1Δ BSD2 cells (MMY157), pep4Δ doa4^C571S RSP5 tul1Δ BSD2 cells (GOY155), or pep4Δ doa4^C571S RSP5 TUL1 bsd2Δ (GOY157) cells transformed with pSNA3-GFP. In A and B, antibodies used for Western blotting are indicated on the left, and the migration of molecular weight standards (kDa) are indicated on the right. (C) Fluorescence and DIC microscopy of bsd2Δ cells (MMY56) transformed with pSNA3-GFP or pGO47. Bar, 2.5 μm.

Figure 3.

The PPAY motif of Sna3 mediates direct binding to Rsp5 WW domains. (A) Schematic diagram of Rsp5 indicating the positions of its C2 domain, three WW domains, and catalytic HECT domain, including amino acid G₅₅₅ important for ubiquitination of MVB cargoes (Katzmann et al., 2004). (B and C) Coomassie-stained gels containing input amounts and glutathione-Sepharose pulldowns of the indicated recombinant fusion proteins expressed in bacteria and purified by affinity isolation. Inputs correspond to 50% of the amount of each recombinant protein used for pulldowns. GST was fused to the N terminus of a segment of Rsp5 containing all three of its WW domains (WW123; pIM2). His₆ was fused to a peptide corresponding to the wild-type C-terminal cytosolic domain of Sna3 (His₆-Sna3^CT WT; pMM143) or to the same peptide sequence containing the P₁₀₆A, P₁₀₇L, Y₁₀₉A, A₁₀₈P, or A₁₀₈Q substitutions (pMM168, pMM154, pMM169, pMM204, or pMM205, respectively). In B and C, the migration of molecular-weight standards (kDa) are indicated on the left.

Figure 4.

Binding of Sna3 to individual WW domains of Rsp5. Coomassie-stained gel containing glutathione-Sepharose pulldowns of the indicated recombinant fusion proteins expressed in bacteria and purified by affinity isolation. GST was fused to the N terminus of a segment of Rsp5 containing each individual wild-type WW domain (WW1, WW2, or WW3; pIM6, pIM10, or pIM8, respectively) or to each WW domain in which the WxxP motif was mutated by replacement of tryptophan with phenylalanine (W/F; pIM35, pIM36, or pIM37 for WW1, WW2, or WW3, respectively) or replacement of both tryptophan and proline with phenylalanine and alanine, respectively (W/F, P/A; pIM38, pIM39, or pIM40 for WW1, WW2, or WW3, respectively). The His₆-Sna3^CT are described in the legend to Figure 3. Migration of molecular weight standards (kDa) are indicated on the left.

Figure 5.

The PPAY motif is required for polyubiquitination and MVB sorting of Sna3-GFP. (A) Western blot analysis of extracts prepared from pep4Δ doa4^C571S sna3Δ cells (MMY153) transformed with wild-type pSNA3-GFP or mutagenized pSNA3-GFP containing the P₁₀₆A, P₁₀₇L, or Y₁₀₉A substitutions (pMM172, pMM99, or pMM171, respectively). Asterisks indicated ubiquitinated forms of Sna3-GFP. Antibodies used for blotting are indicated below each blot, and the migration of molecular weight standards (kDa) are indicated on the right. (B) Fluorescence and DIC microscopy of sna3Δ cells (MMY107) transformed with a high-copy vector encoding DsRed-FYVE (pRS425MET3-DsRed-FYVE; Katzmann et al., 2003) and with wild-type pSNA3-GFP or mutagenized pSNA3-GFP containing the P₁₀₆A, P₁₀₇L, or Y₁₀₉A substitutions (pMM172, pMM99, or pMM171, respectively). Bar, 2.5 μm. (C) Fluorescence and DIC microscopy of FM 4-64–stained rsp5^G555D cells (mvb326) or rsp5Δ cells (GW003) transformed with pSNA3-GFP. Bar, 2.5 μm. Because Rsp5 is required for oleic acid synthesis, which is essential for viability in yeast (Hoppe et al., 2000), rsp5Δ cells were provided supplemental oleic acid in the growth medium.

Figure 6.

Rsp5 is not required for MVB biogenesis. Thin sections (∼70 nm) of wild-type (SEY6210), rsp5^G555D (mvb326), and rsp5Δ (GW003) cells were examined by EM at 80 kV. White scale bars, 100 nm; black scale bars, 500 nm.

Figure 7.

Tomographic analysis of MVB morphology. Two-dimensional cross sections and 3D models derived from 200-nm-thick section tomograms of wild-type (SEY6210; A and B) and rsp5^G555D cells (mvb326; C and D). See corresponding Supplementary Videos. V, vacuole; N, nucleus. Scale bars, 100 nm. The lumenal vesicles in modeled MVBs are color-coded based on diameter (aqua, 18–23 nm; blue, 24–29 nm; bluish purple, 30–35 nm; purple, 36–41). (E) Distribution of vesicle diameters in wild-type and rsp5^G555D cells (n = 330 and 333, respectively) normalized as percent of total. (F) Density of vesicles per unit lumenal MVB volume (nm³) in wild-type and rsp5^G555D cells (p < 0.05, unpaired t test). (G) Vesicle surface area density (nm²) per unit lumenal MVB volume (nm³) in wild-type and rsp5^G555D cells (p = 0.69, unpaired t test). The same data sets for wild-type and rsp5^G555D cells were used to generate the graphs in E–G.