Yeast vacuolar proenzymes are sorted in the late Golgi complex and transported to the vacuole via a prevacuolar endosome-like compartment.

Abstract

We are studying intercompartmental protein transport to the yeast lysosome-like vacuole with a reconstitution assay using permeabilized spheroplasts that measures, in an ATP and cytosol dependent reaction, vacuolar delivery and proteolytic maturation of the Golgi-modified precursor forms of vacuolar hydrolases like carboxypeptidase Y (CPY). To identify the potential donor compartment in this assay, we used subcellular fractionation procedures that have uncovered a novel membrane-enclosed prevacuolar transport intermediate. Differential centrifugation was used to separate permeabilized spheroplasts into 15K and 150K g membrane pellets. Centrifugation of these pellets to equilibrium on sucrose density gradients separated vacuolar and Golgi complex marker enzymes into light and dense fractions, respectively. When the Golgi-modified precursor form of CPY (p2CPY) was examined (after a 5-min pulse, 30-s chase), as much as 30-40% fractionated with an intermediate density between both the vacuole and the Golgi complex. Pulse-chase labeling and fractionation of membranes indicated that p2CPY in this gradient region had already passed through the Golgi complex, which kinetically ordered it between the Golgi and the vacuole. A mutant CPY protein that lacks a functional vacuolar sorting signal was detected in Golgi fractions but not in the intermediate compartment indicating that this corresponds to a post-sorting compartment. Based on the low transport efficiency of the mutant CPY protein in vitro (decreased by sevenfold), this intermediate organelle most likely represents the donor compartment in our reconstitution assay. This organelle is not likely to be a transport vesicle intermediate because EM analysis indicates enrichment of 250-400 nm compartments and internalization of surface-bound 35S-alpha-factor at 15 degrees C resulted in its apparent cofractionation with wild-type p2CPY, indicating an endosome-like compartment (Singer, B., and H. Reizman. 1990. J. Cell Biol. 110:1911-1922). Fractionation of p2CPY accumulated in the temperature sensitive vps15 mutant revealed that the vps15 transport block did not occur in the endosome-like compartment but rather in the late Golgi complex, presumably the site of CPY sorting. Therefore, as seen in mammalian cells, yeast CPY is sorted away from secretory proteins in the late Golgi and transits to the vacuole via a distinct endosome-like intermediate.

Cloning and Subcellular Location of an Arabidopsis Receptor-Like Protein That Shares Common Features with Protein-Sorting Receptors of Eukaryotic Cells

“…The presence of multiple intermediate compartments (early, medial, or late endosomes) between the Golgi, PM, and vacuole/lysosome is well documented in both mammalian and yeast cells (for review, see Pryer et al, 1992;Vida et al, 1993). The transport processes between the Golgi or PM to the endosome are thought to be mediated by CCVs.…”

“…Protein transport from both the PM and TGN to the vacuole/lysosome in yeast and animals proceeds via an inter- (Gruenberg et al, 1989;Vida et al, 1993). The presence of multiple intermediate compartments (early, medial, or late endosomes) between the Golgi, PM, and vacuole/lysosome is well documented in both mammalian and yeast cells (for review, see Pryer et al, 1992;Vida et al, 1993).…”

Vps52p, Vps53p, and Vps54p Form a Novel Multisubunit Complex Required for Protein Sorting at the Yeast Late Golgi

“…A number of sequential budding/fusion reactions take place in the sorting of newly synthesized CPY to the vacuole. CPY bound to its receptor first enters vesicles at the late Golgi that are targeted to fuse with the prevacuolar/ endosomal compartment (PVC) (Vida et al, 1993). Whereas CPY is transported to the vacuole in a second fusion step, its receptor enters retrograde vesicles that bud from the PVC and return to fuse with the late Golgi, thus allowing the receptor to carry out multiple rounds of sorting (Cereghino et al, 1995;Cooper and Stevens, 1996).…”

TheSaccharomyces cerevisiaev-SNARE Vti1p Is Required for Multiple Membrane Transport Pathways to the Vacuole

“…CPY and most other vacuolar enzymes are packaged into transport vesicles, which fuse with the prevacuolar/endosomal compartment (PVC; Vida et al, 1993). The PVC also receives proteins that are endocytosed from the plasma membrane (Vida et al, 1993;Piper et al, 1995). A second fusion step is required for transport from the PVC to the vacuole.…”

“…Genetic screens have identified Ͼ50 genes as being required for transport of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) from the Golgi to the vacuole, including the VPS, PEP, and VAM genes (Jones, 1977;Bankaitis et al, 1986;Rothman and Stevens, 1986;. CPY and most other vacuolar enzymes are packaged into transport vesicles, which fuse with the prevacuolar/endosomal compartment (PVC; Vida et al, 1993). The PVC also receives proteins that are endocytosed from the plasma membrane (Vida et al, 1993;Piper et al, 1995).…”