Abstract
Aggregation of amyloidogenic proteins is associated with several neurodegenerative diseases. Sequestration of misfolded and aggregated proteins into specialized deposition sites may reduce their potentially detrimental properties. Yeast exhibits a distinct deposition site for amyloid aggregates termed “Insoluble PrOtein Deposit (IPOD)”, but nothing is known about the mechanism of substrate recruitment to this site. The IPOD is located directly adjacent to the Phagophore Assembly Site (PAS) where the cell initiates autophagy and the Cytoplasm-to-Vacuole Targeting (CVT) pathway destined for delivery of precursor peptidases to the vacuole. Recruitment of CVT substrates to the PAS was proposed to occur via vesicular transport on Atg9 vesicles and requires an intact actin cytoskeleton and “SNAP (Soluble NSF Attachment Protein) Receptor Proteins (SNARE)” protein function. It is, however, unknown how this vesicular transport machinery is linked to the actin cytoskeleton. We demonstrate that recruitment of model amyloid PrD-GFP and the CVT substrate precursor-aminopeptidase 1 (preApe1) to the IPOD or PAS, respectively, is disturbed after genetic impairment of Myo2-based actin cable transport and SNARE protein function. Rather than accumulating at the respective deposition sites, both substrates reversibly accumulated often together in the same punctate structures. Components of the CVT vesicular transport machinery including Atg8 and Atg9 as well as Myo2 partially co-localized with the joint accumulations. Thus we propose a model where vesicles, loaded with preApe1 or PrD-GFP, are recruited to tropomyosin coated actin cables via the Myo2 motor protein for delivery to the PAS and IPOD, respectively. We discuss that deposition at the IPOD is not an integrated mandatory part of the degradation pathway for amyloid aggregates, but more likely stores excess aggregates until downstream degradation pathways have the capacity to turn them over after liberation by the Hsp104 disaggregation machinery.
Highlights
Protein aggregation occurs through coalescence of misfolded protein species
We found that during their recruitment to the Insoluble PrOtein Deposit (IPOD), amyloid aggregates are linked to transport vesicles that are known to deliver completely unrelated substrates, namely vacuolar peptidase precursors, to a cellular site that is adjacent to the IPOD and is termed Phagophore Assembly Site (PAS) where the cell initiates autophagy
Amyloid aggregates have previously been observed in yeast to accumulate in a particular deposition site termed IPOD [10]
Summary
Protein aggregation occurs through coalescence of misfolded protein species. The cause for acquisition of an aberrant fold can be very diverse ranging from thermal, oxidative or metabolic stress, translational errors, subunit imbalance or mutations to spontaneous or induced conformational rearrangement of intrinsically unstructured proteins such as amyloids [1,2,3]. Yeast as a popular model to study processes related to protein misfolding and aggregation has at least 3 different protein quality control sites for deposition of aggregated proteins, the Juxtanuclear- or Intranuclear Quality control site (JUNQ/INQ), Q-bodies and the IPOD. While JUNQ/INQ and Q-bodies harbor more unstructured, amorphous misfolded proteins, the IPOD is regarded as a specialized deposition site for amyloid aggregates [9,10,11,12]. Insoluble fibrous aggregates with a very high content of β-strands being oriented perpendicularly to the fibril axis. Their occurrence is a hallmark of several fatal neurodegenerative diseases including Parkinson’s Disease, Huntington’s Disease and various prion diseases [2]. It was recently observed that failure of targeting of misfolded proteins to the appropriate deposition site can be associated with cellular toxicity [9,16,17]
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