Peroxisome degradation and housekeeping processes

Group members:  Tim van Zutphen

 

Autophagy (“self-eating”) is the general term to indicate degradation of cytoplasmic components (cytosol, organelles) in eukaryotic cells. This process results in the incorporation and hydrolysis of cell material in vacuoles (in plant and fungal cells) or lysosomes (in animal cells) and its subsequent re-use. Autophagy has long been considered to be predominantly involved in bulk, non-selective turnover of cellular components important for cell survival at nutrient starvation conditions and for cellular housekeeping (e.g. turnover of damaged or unwanted components). However, recent data have demonstrated that autophagy plays a crucial role in development and differentiation and is important in human health and disease (e.g. in ageing and age-associated diseases as well as in cellular defense against invading pathogens).

In recent years a large number of components of the autophagy machinery has been identified. In these studies yeasts are important models because autophagy-deficient yeast mutants are viable. Also peroxisome turn-over is mediated via autophagy-related pathways. Peroxisome numbers can rapidly change in response to changing environmental and/or physiological conditions. For example, the number of peroxisomes rapidly increases upon induction of peroxisome proliferation (e.g. during growth of yeast cells on specific carbon sources). The opposite process, a rapid decrease in peroxisome abundance, can also be induced. Thus, when peroxisomal metabolism is not required anymore, peroxisomes are degraded by vacuoles through autophagy-related pathways. This process is called "pexophagy", and occurs selectively towards peroxisomes. Therefore, it is distinct from non-selective autophagy, which is generally induced by nutrient starvation.

We investigate the molecular mechanisms involved in pexophagy in H. polymorpha, a model organism ideally suited for such studies. In H. polymorpha peroxisomes are massively induced when cells are grown on methanol. Under these conditions the organelles harbor key enzymes of methanol metabolism. Upon a shift of methanol-grown cells to media containing glucose or ethanol, these organelles become redundant and are rapidly and selectively degraded via a process termed macropexophagy.Easy handling of H. polymorpha cells in inducing pexophagy and in genetic manipulation make it possible to study the events of pexophagy in detail.

During macropexophagy, peroxisomes are selectively sequestered one by one by a newly synthesized isolation membrane, which wraps around the peroxisome and forms a double (or multi-)-membrane layered structure termed pexophagosome. The pexophagosome is then delivered to the vacuole, where its outer membrane fuses with the vacuolar membrane, resulting in hydrolysis of the sequestered organelle by vacuolar enzymes.

Research questions in macropexophagy that we currently address are: How does recognition occur of the peroxisome to be degraded?What is the origin of the membranes that sequester peroxisomes from the cytosol? What is the mechanism behind the fusion of sequestered organelles with the vacuole?

Initiation of peroxisome sequestration requires recognition of the organelle to be degraded. Our earlier studies have led to the identification of two peroxisomal membrane proteins that play an important role in the initial steps of macropexophagy (Pex3p and Pex14p).Remarkably, both proteins are peroxins and therefore also required for the biogenesis of peroxisomes. The presence of Pex14p at the peroxisomal membrane is required for recognition of the organelle by the macropexophagy machinery. 

Peroxisome housekeeping processes.

In eukaryotic cells various mechanisms exist that are responsible for the removal of non-functional proteins. We studied how non functional peroxisomal proteins or peroxisomes are removed from the cells. Our data indicated that H. polymorpha peroxisomes contain a Lon protease, Pln, which degrades unfolded and non-assembled peroxisomal matrix proteins. We also observed that whole peroxisomes are constitutively degraded by pexophagy during normal vegetative growth of WT cells.

Deletion of both H. polymorpha PLN and ATG1(in pln.atg1 cells) resulted in a significant decrease in cell viability relative to WT. These data indicate that removal of non-functional peroxisomal proteins/peroxisomes is physiologically very important for the cells.

Because peroxisomes are important organelles in the production of reactive oxygen species (ROS), we analyzed whether defects in peroxisomal housekeeping may lead to ROS accumulation. Peroxisomes harbor hydrogen peroxide producing oxidases together with catalase that is responsible for decomposition of hydrogen peroxide. An imbalance in production and degradation of hydrogen peroxide may result in release of ROS from peroxisomes into the cytosol.

In pln.atg1 cells, but also in cells of pln and atg1 single deletion strains, the intracellular levels of reactive oxygen species was increased relative to WT controls. Most likely this is due to inactivation of peroxisomal catalase, because cytochemical staining experiments revealed that, despite the fact that catalase protein and enzyme activities were normal, not all peroxisomes in pln, atg1 and pln.atg1 cells contained enzymatically active catalase.

In the absence of HpPln or pexophagy unfolded and damaged peroxisomal matrix proteins may accumulate, leading to the formation of sticky protein aggregates that may affect the balance between hydrogen peroxide production and degradation.