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. 2004 Jul 16;279(29):29889-94.
doi: 10.1074/jbc.M404399200. Epub 2004 May 11.

Cargo proteins facilitate the formation of transport vesicles in the cytoplasm to vacuole targeting pathway

Takahiro Shintani  1 , Daniel J Klionsky
Affiliations

Cargo proteins facilitate the formation of transport vesicles in the cytoplasm to vacuole targeting pathway

Takahiro Shintani et al. J Biol Chem. .

Abstract

Selective incorporation of cargo proteins into the forming vesicle is an important aspect of protein targeting via vesicular trafficking. Based on the current paradigm of cargo selection in vesicular transport, proteins to be sorted to other organelles are condensed at the vesicle budding site in the donor organelle, a process that is mediated by the interaction between cargo and coat proteins, which constitute part of the vesicle forming machinery. The cytoplasm to vacuole targeting (Cvt) pathway is an unconventional vesicular trafficking pathway in yeast, which is topologically and mechanistically related to autophagy. Aminopeptidase I (Ape1) is the major cargo protein of the Cvt pathway. Unlike the situation in conventional vesicular transport, precursor Ape1, along with its receptor Atg19/Cvt19, is packed into a huge complex, termed a Cvt complex, independent of the vesicle formation machinery. The Cvt complex is subsequently incorporated into the forming Cvt vesicle. The deletion of APE1 or ATG19 compromised the organization of the pre-autophagosomal structure (PAS), a site that is thought to play a critical role in Cvt vesicle/autophagosome formation. The proper organization of the PAS also required Atg11/Cvt9, a protein that localizes the cargo complex at the PAS. Accordingly, the deletion of APE1, ATG19, or ATG11 affected the formation of Cvt vesicles. These observations suggest a unique concept; in the case of the Cvt pathway, the cargo proteins facilitate receptor recruitment and vesicle formation rather than the situation with most vesicular transport, in which the forming vesicle concentrates the cargo proteins.

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Figures

FIG.1
FIG.1
The Cvt cargo-receptor complex is required for efficient delivery of Atg8 to the vacuole. A, the generation of free GFP from GFP-Atg8 reflects delivery through the Cvt and autophagy pathways. The wild type, atg1Δ, and pep4Δ cells expressing GFP-Atg8 (YTS187, YTS188, and YTS189, respectively) were grown in SCD (rich medium; growing conditions) to A600 = 1.0 and starved in SD(-N) for 3 h. Cell lysates equivalent to A600 = 0.1 unit of cells were subjected to immunoblot analysis with anti-Ape1 antiserum and anti-GFP antibody. B, delivery of Atg8 to the vacuole was blocked or extensively delayed in the absence of Ape1, Atg19, and Atg11. The atg1ts, atg1ts ape1Δ, atg1ts atg19Δ, and atg1ts atg11Δ cells expressing GFP-Atg8 (YTS201, YTS203, YTS202, and YTS204, respectively) were grown in SCD to A600 = 1.0 at a non-permissive temperature of 37 °C. After treatment with or without 0.2 μg/ml rapamycin (Rap) for 10 min at 37 °C, the cells were transferred to a permissive temperature of 30 °C and incubated for the indicated times. Ape1 and GFP-Atg8 were analyzed by immunoblotting as indicated in A. The positions of precursor and mature Ape1 (mApe1) and of full-length GFP-Atg8 and free GFP are indicated. The asterisk marks a nonspecific cross-reacting band.
FIG.2
FIG.2
Quantification of cells containing pre-autophagosomal structures. The wild type, ape1Δ, atg11Δ, and atg19Δ cells expressing GFP-Atg8 (YTS187, YTS190, YTS191, and YTS192), GFP-Atg2 (YTS193, YTS194, YTS195, and YTS196), GFP-Atg1 (YTS197, YTS198, YTS199, and YTS200), or Atg20-YFP (YTS180, YTS184, YTS185, and TYY014) were grown in SCD (rich medium; growing conditions) to A600 = 1.0 and starved in SD(-N) for 1 h. For each strain, 150-250 cells containing fluorescent punctate dots were quantified by fluorescence microscopy. Error bars indicate the S.D. of three independent experiments. The chromosomal VPS38 gene was deleted in the cells expressing Atg20-YFP to eliminate punctate localization at the endosome (see text for details). Punctate localization of GFP-Atg8, GFP-Atg2, GFP-Atg1, and Atg20-YFP to the PAS was greatly diminished in the absence of the Cvt cargo-receptor complex under growing conditions, but not under starvation conditions.
FIG.3
FIG.3
Atg9 cycling requires the Cvt cargo-receptor complex under growing conditions. The atg1Δ, atg1Δ ape1Δ, and atg1Δ atg19Δ cells expressing Atg9-YFP (FRY138, YTS136, and YTS135, respectively) were grown in SCD (rich medium; growing conditions) and then shifted to SD(-N) (starvation conditions) and observed by fluorescence microscopy. Atg9 normally transits to the PAS and is retrieved from this site in an Atg1-dependent manner. Delivery to the PAS was defective in the absence of the Cvt cargo-receptor complex.
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