Routing misfolded proteins through the multivesicular body (MVB) pathway protects against proteotoxicity

Abstract

The secretory pathway maintains multiple quality control checkpoints. Initially, endoplasmic reticulum-associated degradation pathways monitor protein folding to retain and eliminate aberrant products. Despite its broad client range, some molecules escape detection and traffic to Golgi membranes. There, a poorly understood mechanism termed Golgi quality control routes aberrant proteins for lysosomal/vacuolar degradation. To better understand Golgi quality control, we examined the processing of the obligate substrate Wsc1p. Misfolded Wsc1p does not use routes of typical vacuolar membrane proteins. Instead, it partitions into intralumenal vesicles of the multivesicular body (MVB) pathway, mediated by the E3 ubiquitin ligase Rsp5p. Its subsequent transport to the vacuolar lumen is essential for complete molecule breakdown. Surprisingly, the transport mode plays a second crucial function in neutralizing potential substrate toxicity. Eliminating the MVB sorting signal diverted molecules to the vacuolar limiting membrane, resulting in the generation of toxic by-products. These data demonstrate a new role of the MVB pathway in protein quality control.

FIGURE 1.

Wsc1* localizes to the vacuolar lumen. A, schematic representation of Wsc1*. The L63R mutation is indicated by the asterisk, and the position of the HA epitope tag is shown in black. S.S., signal sequence; TM, transmembrane domain; Cyt, cytoplasmic domain. B, Wsc1* in wild type and Δpep4 cells was localized by indirect immunofluorescence using anti-HA monoclonal antibody and Alexa Fluor 488 goat anti-mouse antibody (green channel). Nuclear DNA was stained by DAPI to indicate positions of nuclei (blue channel). Visualization was performed using confocal and DIC microscopy as indicated. Scale bar, 5 μm. C, Wsc1* localization in Δpep4 cells as described in B. A z-series was captured, with the middle plane, its corresponding DIC image, and merged images shown in a–c. d–k show individual z-stacks of the series from top to bottom. Scale bars, 1 μm. D, wild type and Δpep4 cells were processed as in B and bound with anti-V-ATPase (60-kDa subunit) antibody as a vacuolar membrane marker followed by Alexa Fluor 488 goat anti-mouse antibody. Cells were visualized by confocal and DIC microscopy. Scale bar, 5 μm.

FIGURE 2.

ESCRT mutants alter trafficking and degradation patterns of Wsc1*. A, wild type and Δpep4, Δvps27, and Δpep4Δvps27 mutants expressing Wsc1* were processed for indirect immunofluorescence, as described in the legend to Fig. 1B. Cells were visualized by confocal and DIC microscopy. Scale bar, 5 μm. B, a–c display the middle plane of a z-series as described for Fig. 1B captured from Δpep4Δvps27 cells expressing Wsc1*. d–o display the z-stack series from top to bottom. Scale bars, 1 μm. C, Wsc1* expression in wild type and Δpep4, Δvps27, and Δpep4Δvps27 mutants was analyzed by immunoblotting. Membranes were probed with anti-HA antibody (top), stripped, and reprobed with anti-PGK antibody as the loading control (bottom).

FIGURE 3.

Wsc1* requires Rsp5p and Doa4p for entry into the MVB pathway. A, wild type, Δpep4, rsp5-1, and Δpep4 rsp5-1 cells expressing Wsc1* were grown to log phase and shifted to 37 °C for 1 h. Cell preparation and image acquisition were performed as described in the legend to Fig. 1B. Scale bar, 5 μm. B, indirect immunofluorescence of wild type, Δpep4, Δdoa4, and Δpep4Δdoa4 strains expressing Wsc1*. Scale bar, 5 μm. C, Western blot analysis of wild type, Δpep4, rsp5-1, and Δpep4 rsp5-1 cells expressing Wsc1* after a 1-h shift to 37 °C. D, Wsc1* expression in wild type, Δpep4, Δdoa4, and Δpep4Δdoa4 cells was analyzed by immunoblotting as described in the legend to Fig. 2C.

FIGURE 4.

A Wsc1* ubiquitination-deficient variant fails to enter the MVB pathway. Wsc1*-6R localization in wild type (A) and Δpep4 cells (B). Images in a–c are from the middle plane of a z-series displaying Wsc1* (HA), vacuoles (DIC), and their merged images. d–m (A) and d–k (B) show a series of z-stacks from top to bottom. Scale bars, 1 μm. C, Wsc1* and Wsc1*-6R expression in wild type and Δpep4 cells analyzed by immunoblotting using the anti-HA antibody.

FIGURE 5.

Wsc1* degradation intermediates are stable in Δvps27 cells. A, cycloheximide chase analysis of wild type and Δvps27 cells expressing Wsc1*. Chase times following the cycloheximide addition are shown above the blots. Wsc1* was detected using anti-HA antibodies (top). Anti-PGK antibody was used to probe the stripped blot as a loading control (bottom). B, cycloheximide chase analysis was performed on wild type and Δpep4 cells expressing Wsc1*-6R as described in A.

FIGURE 6.

Accumulation of Wsc1* fragments causes toxicity. A, wild type, Δpep4, and Δvps27 cells containing (P_GAL1)Wsc1* or (P_GAL1)CPY* grown in raffinose media were spotted as 10-fold serial dilutions onto glucose (promoter-repressed) and galactose (promoter-activated) medium plates and incubated at 30 °C for 2 and 3 days, respectively. B, wild type and Δpep4 cells containing the control vector, (P_GAS1)Wsc1*, or (P_GAS1)Wsc1*-6R were spotted on selective synthetic plates and incubated at 30 °C for 2 days. C, growth analysis of wild type and Δpep4 cells containing the empty vector (pRS315), (P_GAS1)Wsc1*, or (P_GAS1)Wsc1*-6R. Cells were grown in selective synthetic media to log phase and diluted to 0.1 A/ml. Growth was measured as 0.1 A₆₀₀/ml readings from cultures at regular intervals over 10 h. The data plotted reflect three independent experiments with the mean ± S.D. (error bars) indicated. *, p < 0.01, Student's t test. D, membranes prepared from wild type and Δpep4 cells expressing (P_GAS1)Wsc1*-6R were treated with 0.1 m sodium carbonate, pH 11.0, for 30 min on ice. A portion was reserved as total (T), and the remaining was subjected to centrifugation at 100,000 × g. Supernatant (S) and membrane pellet (P) fractions were collected and analyzed by immunoblotting. Wsc1*-6R was detected using anti-HA antibody. Kar2p and Sec61p serve as soluble and integral membrane protein controls, respectively.

FIGURE 7.

Wsc1*-6R degradation by-products disrupt vacuolar membrane morphology. Wild type cells with an empty vector (pRS315), (P_GAS1)Wsc1*, or (P_GAS1)Wsc1*-6R and Δpep4 cells expressing (P_GAS1)Wsc1* or (P_GAS1)Wsc1*-6R were grown to log phase and stained with FM4-64 at 30 °C. Cell imaging was performed using confocal and DIC microscopy. Scale bar, 5 μm.

FIGURE 8.

Model of the MVB-dependent pathway for the transport of misfolded Wsc1p. A, normally, misfolded Wsc1p exits the ER and transits through the Golgi apparatus. It is next sorted to the MVB pathway and degraded in its entirety within the vacuolar lumen. This mechanism requires the ubiquitin ligase Rsp5p and ubiquitination of the Wsc1p cytoplasmic domain. B, misfolded GQC substrates missorted to the vacuolar limiting membrane. Degradation proceeds but is constrained by the membrane, leading to partial degradation products still integrated in the membrane. These aberrant products disrupt vacuolar membranes, which can lead to cell death.