Suresh Subramani

A unified nomenclature for yeast autophagy-related genes

The authors would like to thank Drs. Jan A.K.W. Kiel, Ida J. van der Klei, Beth Levine, Fulvio Reggiori, and Takahiro Shintani for helpful comments on the manuscript, and the many researchers in the yeast field who have agreed to changes in the standard names of various genes.

A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase.

Several peroxisomal proteins do not contain the previously identified tripeptide peroxisomal targeting signal (PTS) at their carboxy‐termini. One such protein is the peroxisomal 3‐ketoacyl CoA thiolase, of which two types exist in rat [Hijikata et al. (1990) J. Biol. Chem., 265, 4600–4606]. Both rat peroxisomal thiolases are synthesized as larger precursors with an amino‐terminal prepiece of either 36 (type A) or 26 (type B) amino acids, that is cleaved upon translocation of the enzyme into the peroxisome. The prepieces are necessary for import of the thiolases into peroxisomes because expression of an altered cDNA encoding only the mature thiolase, which lacks any prepiece, results in synthesis of a cytosolic enzyme. When appended to an otherwise cytosolic passenger protein, the bacterial chloramphenicol acetyltransferase (CAT), the prepieces direct the fusion proteins into peroxisomes, demonstrating that they encode sufficient information to act as peroxisomal targeting signals. Deletion analysis of the thiolase B prepiece shows that the first 11 amino acids are sufficient for peroxisomal targeting. We conclude that we have identified a novel PTS that functions at amino‐terminal or internal locations and is distinct from the C‐terminal PTS. These results imply the existence of two different routes for targeting proteins into the peroxisomal matrix.

Firefly luciferase as a tool in molecular and cell biology

The unique properties of firefly luciferase and the cloning of the gene for this enzyme have spawned a number of novel applications of this protein. We summarize a few of these applications including its use as a reporter gene, as a model for the study of protein import into peroxisomes, and as a component of a heterologous gene expression system.

Peroxisomal protein import is conserved between yeast, plants, insects and mammals.

We have previously demonstrated that firefly luciferase can be imported into peroxisomes of both insect and mammalian cells. To determine whether the process of protein transport into the peroxisome is functionally similar in more widely divergent eukaryotes, the cDNA encoding firefly luciferase was expressed in both yeast and plant cells. Luciferase was translocated into peroxisomes in each type of organism. Experiments were also performed to determine whether a yeast peroxisomal protein could be transported to peroxisomes in mammalian cells. We observed that a C‐terminal segment of the yeast (Candida boidinii) peroxisomal protein PMP20 could act as a peroxisomal targeting signal in mammalian cells. These results suggest that at least one mechanism of protein translocation into peroxisomes has been conserved throughout eukaryotic evolution.

Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation

Evidence is presented for an unconventional protein secretion pathway that is conserved from yeast to Dictyostelium discoideum in which Acb1 may be sequestered into autophagosomal vesicles, which then fuse (either directly or indirectly) with the plasma membrane (see also the companion paper from Duran et al. in this issue).

PpAtg30 tags peroxisomes for turnover by selective autophagy.

Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation. The mechanism of selective peroxisome turnover by autophagy-related processes (pexophagy), termed micropexophagy and macropexophagy, is unknown. We show how a Pichia pastoris protein, PpAtg30, mediates peroxisome selection during pexophagy. It is necessary for pexophagy, but not for other selective and nonselective autophagy-related processes. It localizes at the peroxisome membrane via interaction with peroxins, and during pexophagy it colocalizes transiently at the preautophagosomal structure (PAS) and interacts with the autophagy machinery. PpAtg30 is required for formation of pexophagy intermediates, such as the micropexophagy apparatus (MIPA) and the pexophagosome (Ppg). During pexophagy, PpAtg30 undergoes multiple phosphorylations, at least one of which is required for pexophagy. PpAtg30 overexpression stimulates pexophagy even under peroxisome-induction conditions, impairing peroxisome biogenesis. Therefore, PpAtg30 is a key player in the selection of peroxisomes as cargo and in their delivery to the autophagy machinery for pexophagy.

The Human Peroxisomal Targeting Signal Receptor, Pex5p, Is Translocated into the Peroxisomal Matrix and Recycled to the Cytosol

Peroxisomal targeting signals (PTSs) are recognized by predominantly cytosolic receptors, Pex5p and Pex7p. The fate of these PTS receptors following their interactions on the peroxisomal membrane with components of docking and putative translocation complexes is unknown. Using both novel and multiple experimental approaches, we show that human Pex5p does not just bind cargo and deliver it to the peroxisome membrane, but participates in multiple rounds of entry into the peroxisome matrix and export to the cytosol independent of the PTS2 import pathway. This unusual shuttling mechanism for the PTS1 receptor distinguishes protein import into peroxisomes from that into most other organelles, with the exception of the nucleus.

Overexpression of Pex15p, a phosphorylated peroxisomal integral membrane protein required for peroxisome assembly in S.cerevisiae, causes proliferation of the endoplasmic reticulum membrane

We have cloned PEX15 which is required for peroxisome biogenesis in Saccharomyces cerevisiae. pex15Δ cells are characterized by the cytosolic accumulation of peroxisomal matrix proteins containing a PTS1 or PTS2 import signal, whereas peroxisomal membrane proteins are present in peroxisomal remnants. PEX15 encodes a phosphorylated, integral peroxisomal membrane protein (Pex15p). Using multiple in vivo methods to determine the topology, Pex15p was found to be a tail‐anchored type II (Ncyt–Clumen) peroxisomal membrane protein with a single transmembrane domain near its carboxy‐terminus. Overexpression of Pex15p resulted in impaired peroxisome assembly, and caused profound proliferation of the endoplasmic reticulum (ER) membrane. The lumenal carboxy‐terminal tail of Pex15p protrudes into the lumen of these ER membranes, as demonstrated by its O‐glycosylation. Accumulation in the ER was also observed at an endogenous expression level when Pex15p was fused to the N‐terminus of mature invertase. This resulted in core N‐glycosylation of the hybrid protein. The lumenal C‐terminal tail of Pex15p is essential for targeting to the peroxisomal membrane. Furthermore, the peroxisomal membrane targeting signal of Pex15p overlaps with an ER targeting signal on this protein. These results indicate that Pex15p may be targeted to peroxisomes via the ER, or to both organelles.

How proteins penetrate peroxisomes

Three decades after the unobtrusive debut of the peroxisome as a distinct subcellular organelle, biologists are paying attention to the special bag of tricks eukaryotic cells use to entice peroxisomal proteins from their site of synthesis in the cytosol to the peroxisome. In this minireview, we highlight some of the recent findings that have emerged, emphasize their significance, and contrast them with aspects of protein import into other subcellular destinations. Peroxisomal Targeting Signals The Beginning, the Middle, and the End Genetic and biochemical evidence has underscored the conservation of peroxisomal targeting signals (PTSs) from yeast to humans and led to the elucidation of at least two pathways for the transport of proteins to the peroxisome matrix (lumen). Each of these pathways is dependent on the molecular recognition of a specific PTS by its cognate receptor, which then hands off the protein to a putative translocation machinery housed in the peroxisome membrane. PTSl is a conserved C-terminal tripeptide (SKL or a variant) that constitutes the major targeting signal for proteins destined for the peroxisome matrix. In contrast, PTS2 is a conserved N-terminal nonapeptide (R/K)(L/V/I) (X)5(H/Q)(L/A) used by a smaller subset of peroxisomal matrix proteins. Other internally located PTSs have been described but remain poorly characterized (Purdue and Lazarow, 1994). Peroxisomal membrane proteins do not possess either PTSl or PTS2 sequences but are endowed instead with PTSs that have been defined only as fairly large internal segments of peroxisomal membrane proteins (Purdue and Lazarow, 1994). Despite the fact that all these PTSs are known to be necessary and sufficient for targeting to peroxisomes, a novel twist (addressed later) is the recent discovery that polypeptide chains devoid of a PTS can hitch a ride into peroxisomes by association with subunits that contain a PTS (Glover et al., 1994; McNew and Goodman, 1994). Omnipresent PTS Receptors Yeast and human cells selectively deficient in the PTS1 or PTS2 import pathway (or both) have been instrumental in the identification of PTS receptors. The earliest mutant discovered to be selectively compromised in the PTSl pathway alone was the pas8 mutant of Pichia pastoris. The protein, Pas8p, is tightly associated with the cytosolic face of the peroxisome membrane and is the PTSl receptor (PTSl R) (Terlecky et al., 1995; Figure 1). Minireview

THE PICHIA PASTORIS PEROXISOMAL PROTEIN PAS8P IS THE RECEPTOR FOR THE C-TERMINAL TRIPEPTIDE PEROXISOMAL TARGETING SIGNAL

The peroxisomal targeting signal 1 (PTS1), consisting of a C‐terminal tripeptide (SKL and variants), directs polypeptides to the peroxisome matrix in evolutionarily diverse organisms. Previous studies in the methylotrophic yeast Pichia pastoris identified a 68 kDa protein, PAS8p, as a potential component of the PTS1 import machinery. We now report several new properties of this molecule which, taken together, show that it is the peroxisomal PTS1 receptor. (i) PAS8p is localized to and tightly associated with the cytoplasmic side of the peroxisomal membrane, (ii) peroxisomes of wild‐type, but not of pas8 delta (null) mutant, P.pastoris cells bind a PTS1‐containing peptide (CRYHLKPLQSKL), (iii) CRYHLKPLQSKL can be cross‐linked to PAS8p after binding at the peroxisome membrane and (iv) purified PAS8p binds CRYHLKPLQSKL with high affinity (nanomolar dissociation constant). In addition, the tetratricopeptide repeat (TPR) domain of PAS8p is identified as the PTS1 binding region.