The yeast lysosome-like vacuole: endpoint and crossroads

Abstract

Fungal vacuoles are acidic organelles with degradative and storage capabilities that have many similarities to mammalian lysosomes and plant vacuoles. In the past several years, well-developed genetic, genomic, biochemical and cell biological tools in S. cerevisiae have provided fresh insights into vacuolar protein sorting, organelle acidification, ion homeostasis, autophagy, and stress-related functions of the vacuole, and these insights have often found parallels in mammalian lysosomes. This review provides a broad overview of the defining features and functions of S. cerevisiae vacuoles and compares these features to mammalian lysosomes. Recent research challenges the traditional view of vacuoles and lysosomes as simply the terminal compartment of biosynthetic and endocytic pathways (i.e. the "garbage dump" of the cell), and suggests instead that these compartments are unexpectedly dynamic and highly regulated.

Figure 1. Vacuolar morphology is sensitive to growth conditions

The vacuole can change in volume and vesicle number depending on extracellular condtions. Top: Under normal conditions, the cell has two to three medium-sized vacuoles. In hypo-osmotic media, a single large vacuole takes up most of the cell. Upon hyper-osmotic shock, the vacuole fragments into multiple small lobes. Bottom: Superimposed images of fluorescently stained vacuoles on yeast cells viewed under Nomarski optics. Vacuoles were visualized using a C-terminally GFP-tagged V-ATPase subunit (Vma2p-GFP) during log-phase growth under hypotonic conditions (synthetic complete medium), normal conditions (YEPD, buffered to pH 5 (100 mM added salt)), and hypertonic conditions (YEPD, buffered to pH 5 with 1 M NaCl added).

Figure 2. Trafficking Pathways to and from the Vacuole

Multiple vesicular pathways deliver proteins to and from the vacuole. Resident vacuolar proteins are sent to the vacuole by several biosynthetic pathways, while proteins targeted for degradaton may be sent via endocytosis or autophagy. Biosynthetic pathways: The ALP pathway delivers cargo from the late Golgi to the vacuole. The CPY pathway also starts from the late Golgi, but traverses multivesicular bodies (MVBs) before reaching the vacuole. The CVT pathway delivers biosynthetic cargo from the cytosol to the vacuole in a process that has common components with autophagy. Trafficking pathways away from the vacuole may recycle membrane and proteins to the endocytic or CPY pathways. Degradative pathways: Endocytosis transports both soluble and membrane-bound cargo from the plasma membrane and extracellular space to the vacuole. Cargo progresses from early endosomes to late endosomes (multivesicular bodies) before reaching the vacuole. In macroautophagy, vesicles called autophagosomes engulf cytosolic material for degradation at the vacuole. In microautophagy, the vacuolar membrane itself forms invaginations which form vesicles that are then degraded at the vacuole.

Figure 3. The Vacuolar H⁺-ATPase

The vacuolar H^+-ATPase (V-ATPase) is a proton pump on the vacuolar membrane composed of a membrane-bound V₀ sector and a cytosolic V₁ sector. ATP hydrolysis in the V₁ sector drives the translocation of protons through the V₀ sector.

Figure 4. Constitutive Functions of The Yeast Vacuole

The vacuole is responsible for multiple constitutive processes: 1) degradation, 2) storage, 3) buffering, and 4) detoxification. 1) Degradation: The vacuole is highly enriched for hydrolases (yellow circles) that break down cargo delivered via multiple trafficking pathways to the vacuole. 2) Storage: The vacuolar membrane contains multiple transporters (blue polygons) that import amino acids, ions, and metals. Vacuolar acidification by the vacuolar H⁺ ATPase (V₁ and V₀, green) is required for proper maturation of hydrolases and for establishing the proton gradient that drives many transporters. 3) Buffering: The vacuolar membrane also contains transporters that recycle amino acids, ions, and metals to the cytosol. The combined activity of importers and exporters is important for ion homeostasis and amino acid recycling. The vacuole is also the main storage site for polyphosphate which buffers cations and is a source for cellular phosphate, 4) Detoxification: The vacuole also sequesters toxic metals and potentially harmful metabolic bi-products via ABC transporters.

Figure 5. Stress responses

Panel A: Upon nutrient deprivation, multiple autophagic pathways deliver specific and non-specific cargo to the vacuole. Macroautophagy is the non-specific degradation of cytosolic proteins and organelles. Autophagic membranes originate from pre-autosomal structures (PAS) that enclose cytosolic cargo, forming autophagosomes, which are then delivered to the vacuole. In microautophagy, vacuolar membranes invaginate and directly engulf cargo for degradation. Piecemeal microautophagy of the nucleus (PMN) is a type of autophagy that specifically degrades nuclear compartments, while pexophagy degrades peroxisomes via two mechanisms similar to macroautophagy and microautophagy. Panel B: Upon salt stress, vacuoles fragment into multiple tiny vesicles. This requires increased production of PI_3,5P₂ by the Fab1p lipid kinase. Calcium is released from vacuolar stores by Yvc1p, activating calcineurin and the Crz1p transcription factor. This leads to the increased production of the Ena1p Na⁺/Li⁺ pump at the plasma membrane and the Pho89p Na⁺/Pi⁺ symporter at the vacuole.