Dual role of ER stress in response to metabolic co-targeting and radiosensitivity in head and neck cancer cells.

Arginine deprivation therapy (ADT) is a new metabolic targeting approach with high therapeutic potential for various solid cancers. Combination of ADT with low doses of the natural arginine analog canavanine effectively sensitizes malignant cells to irradiation. However, the molecular mechanisms determining the sensitivity of intrinsically non-auxotrophic cancers to arginine deficiency are still poorly understood. We here show for the first time that arginine deficiency is accompanied by global metabolic changes and protein/membrane breakdown, and results in the induction of specific, more or less pronounced (severe vs. mild) ER stress responses in head and neck squamous cell carcinoma (HNSCC) cells that differ in their intrinsic ADT sensitivity. Combination of ADT with canavanine triggered catastrophic ER stress via the eIF2α-ATF4(GADD34)-CHOP pathway, thereby inducing apoptosis; the same signaling arm was irrelevant in ADT-related radiosensitization. The particular strong supra-additive effect of ADT, canavanine and irradiation in both intrinsically more and less sensitive cancer cells supports the rational of ER stress pathways as novel target for improving multi-modal metabolic anti-cancer therapy.

Indospicine combined with arginine deprivation triggers cancer cell death via caspase-dependent apoptosis.

Arginine-deprivation therapy is a rapidly developing metabolic anticancer approach. To overcome the resistance of some cancer cells to this monotherapy, rationally designed combination modalities are needed. In this report, we evaluated for the first time indospicine, an arginine analogue of Indigofera plant genus origin, as potential enhancer compound for the metabolic therapy that utilizes recombinant human arginase I. We demonstrate that indospicine at low micromolar concentrations is selectively toxic for human colorectal cancer cells only in the absence of arginine. In arginine-deprived cancer cells indospicine deregulates some prosurvival pathways (PI3K-Akt and MAPK) and activates mammalian target of rapamycin, exacerbates endoplasmic reticulum stress and triggers caspase-dependent apoptosis, which is reversed by the exposure to translation inhibitors. Simultaneously, indospicine is not degraded by recombinant human arginase I and does not inhibit this arginine-degrading enzyme at its effective dose. The obtained results emphasize the potential of arginine structural analogues as efficient components for combinatorial metabolic targeting of malignant cells.

Effects of Arginine and Its Deprivation on Human Glioblastoma Physiology and Signaling.

The observations that numerous cancers are characterized by impairment in arginine synthesis and that deficit of exogenous arginine specifically affects their growth and viability are the ground for arginine deprivation-based anticancer treatment strategy. This review addresses molecular mechanisms of the human glioblastoma cell response to arginine deprivation. Our earlier studies have shown that arginine deprivation specifically impairs glioblastoma cell motility, adhesion and invasiveness. These changes were evoked by alterations in the actin cytoskeleton organization resulting from a decreased arginylation of ß-actin isoform. Moreover, deficit of arginine induces prolonged endoplasmic reticulum (ER) stress and activation of the unfolded protein response, not leading, however, to a massive apoptosis in glioblastoma cells. Our current research indicates that cell death could be augmented by other compounds such as modulators of ER stress, for example arginine analogue of plant origin, canavanine. Implication of these studies on the development of new anti-glioma metabolic therapeutic modalities are discussed.

Nitric oxide donor augments antineoplastic effects of arginine deprivation in human melanoma cells.

Anticancer therapy based on recombinant arginine-degrading enzymes has been proposed for the treatment of several types of malignant cells deficient in arginine biosynthesis. One of the predicted side effects of such therapy is restricted bioavailability of nitric oxide as arginine catabolic product. Prolonged NO limitation may lead to unwanted disturbances in NO-dependent vasodilation, cardiovascular and immune systems. This problem can be overcome by co-supplementation with exogenous NO donor. However, NO may potentially counteract anticancer effects of therapy based on arginine deprivation. In this study, we evaluate for the first time the effects of an exogenous NO donor, sodium nitroprusside, on viability and metastatic properties of two human melanoma cell lines SK-MEL-28 and WM793 under arginine-deprived conditions. It was revealed that NO did not rescue melanoma cells from specific effects evoked by arginine deprivation, namely decreased viability and induction of apoptosis, dramatically reduced motility, invasiveness and clonogenic potential. Moreover, sodium nitroprusside co-treatment augmented several of these antineoplastic effects. We report that a combination of NO-donor and arginine deprivation strongly and specifically impaired metastatic behavior of melanoma cells. Thus, sodium nitroprusside can be considered as an adjuvant for the more efficient treatment of malignant melanoma and possibly other tumors with arginine-degrading enzymes.

Arginine starvation in colorectal carcinoma cells: Sensing, impact on translation control and cell cycle distribution.

Tumor cells rely on a continued exogenous nutrient supply in order to maintain a high proliferative activity. Although a strong dependence of some tumor types on exogenous arginine sources has been reported, the mechanisms of arginine sensing by tumor cells and the impact of changes in arginine availability on translation and cell cycle regulation are not fully understood. The results presented herein state that human colorectal carcinoma cells rapidly exhaust the internal arginine sources in the absence of exogenous arginine and repress global translation by activation of the GCN2-mediated pathway and inhibition of mTOR signaling. Tumor suppressor protein p53 activation and G1/G0 cell cycle arrest support cell survival upon prolonged arginine starvation. Cells with the mutant or deleted TP53 fail to stop cell cycle progression at defined cell cycle checkpoints which appears to be associated with reduced recovery after durable metabolic stress triggered by arginine withdrawal.

Arginine deprivation affects glioblastoma cell adhesion, invasiveness and actin cytoskeleton organization by impairment of ß-actin arginylation.

A deficit of exogenous arginine affects growth and viability of numerous cancer cells. Although arginine deprivation-based strategy is currently undergoing clinical trials, molecular mechanisms of tumor cells' response to arginine deprivation are not yet elucidated. We have examined effects of arginine starvation on cell motility, adhesion and invasiveness as well as on actin cytoskeleton organization of human glioblastoma cells. We observed for the first time that arginine, but not lysine, starvation affected cell morphology, significantly inhibited their motility and invasiveness, and impaired adhesion. No effects on glia cells were observed. Also, arginine deprivation in glioblastoma evoked specific changes in actin assembly, decreased ß-actin filament content, and affected its N-terminal arginylation. We suggest that alterations in organization of ß-actin resulted from a decrease of its arginylation could be responsible for the observed effects of arginine deprivation on cell invasiveness and migration. Our data indicate that arginine deprivation-based treatment strategies could inhibit, at least transiently, the invasion process of highly malignant brain tumors and may have a potential for combination therapy to extend overall patient survival.

Recombinant arginine-degrading enzymes in metabolic anticancer therapy and bioanalytics.

Tumor cells often exhibit specific metabolic defects due to the aberrations in oncogene-dependent regulatory and/or signaling pathways that distinguish them from normal cells. Among others, many malignant cells are deficient in biosynthesis of certain amino acids and concomitantly exhibit elevated sensitivity to deprivation of these amino acids. Although the underlying causes of such metabolic changes are still not fully understood, this feature of malignant cells is exploited in metabolic enzymotherapies based on single amino acid, e.g., arginine, deprivation. To achieve efficient arginine depletion in vivo, two recombinant enzymes, bacterial arginine deiminase and human arginase I have been evaluated and are undergoing further development. This review is aimed to summarize the current knowledge on the application of arginine-degrading enzymes as anticancer agents and as bioanalytical tools for arginine assays. The problems that have to be solved to optimize this therapy for clinical application are discussed.

Three-dimensional environment renders cancer cells profoundly less susceptible to a single amino acid starvation.

Increased amino acid requirement of malignant cells is exploited in metabolic antitumor therapy, e.g., enzymotherapies based on arginine or methionine deprivation. However, studies on animal models and clinical trials revealed that solid tumors are much less susceptible to single amino acid starvation than could be expected from the in vitro data. We conducted a comparative analysis of the response of several tumor cell lines to single amino acid starvation in 2-D monolayer versus 3-D spheroid culture. We revealed for the first time that in comparison with monolayer culture tumor cells, spheroids are much less susceptible to the deprivation of individual amino acids (i.e., arginine, leucine, lysine or methionine). Accordingly, even after prolonged (up to 10 days) starvation, spheroid cells could readily resume proliferation when appropriate amino acid was resupplemented. In the case of arginine deprivation, similar apoptosis induction was detected both in 2-D and 3-D culture, suggesting that this process does not determine the level of tumor cell sensitivity to this kind of treatment. It was also observed that spheroids much better mimic the in vivo ability of tumor cells to utilize citrulline as arginine precursor for growth in amino acid deficient environment. We conclude that 3-D spheroid culture better reflects in vivo tumor cell response to single amino acid starvation than 2-D monolayer culture and should be used as an integral model in the studies of this type of antitumor metabolic targeting.

Stable overproducer of hepatitis B surface antigen in the methylotrophic yeast Hansenula polymorpha due to multiple integration of heterologous auxotrophic selective markers and defect in peroxisome biogenesis.

Two methods of multicopy integrant selection in the methylotrophic yeast Hansenula polymorpha based on the use of heterologous yeast auxotrophic genes have been used to isolate effective overproducers of hepatitis B surface antigen (HBsAg). One selection marker was described earlier for this yeast, the Saccharomyces cerevisiae URA3 gene, whereas the second selection marker was developed by us, the Pichia pastoris ADE1 gene with shortened native promoter. Sequential use of both selection markers produced stable transformants containing up to 30 integration cassettes with HBsAg gene. Deletion of PEX3 gene coding for peroxine involved in the early step of peroxisome formation substantially increased the production of HBsAg in glucose medium as compared to the parental strain. Maximal production of HBsAg in Δpex3 strain was nearly 8-9% of the total cell protein.

Single amino acid arginine starvation efficiently sensitizes cancer cells to canavanine treatment and irradiation.

Single amino acid arginine deprivation is a promising strategy in modern metabolic anticancer therapy. Its potency to inhibit tumor growth warrants the search for rational chemo- and radio-therapeutic approaches to be co-applied. In this report, we evaluated, for the first time, the efficacy of arginine deprivation as anticancer therapy in three-dimensional (3D) cultures of human tumor cells, and propose a new combinatorial metabolic-chemo-radio-treatment regime based on arginine starvation, low doses of arginine natural analog canavanine and irradiation. A sophisticated experimental setup was designed to evaluate the impact of arginine starvation on four human epithelial cancer cell lines in 2D monolayer and 3D spheroid culture. Radioresponse was assessed in colony formation assays and by monitoring spheroid regrowth probability following single dose irradiation using a standardized spheroid-based test platform. Surviving fraction at 2 Gy (SF(2Gy)) and spheroid control dose(50) (SCD(50) ) were calculated as analytical endpoints. Cancer cells in spheroids are much more resistant to arginine starvation than in 2D culture. Spheroid volume stagnated during arginine deprivation, but even after 10 days of starvation, 100% of the spheroids regrew. Combination treatment, however, was remarkably efficient. In particular, pretreatment of cancer cells with the arginine-degrading enzyme arginase combined with or without low concentration of canavanine substantially enhanced cell radioresponse reflected by a loss in spheroid regrowth probability and SCD(50) values reduced by a factor of 1.5-3. Our data strongly suggest that arginine withdrawal alone or in combination with canavanine is a promising antitumor strategy with potential to enhance cancer cure by irradiation.

Overexpression of (His)6-tagged human arginase I in Saccharomyces cerevisiae and enzyme purification using metal affinity chromatography.

Arginase (EC 3.5.3.1; L-arginine amidinohydrolase) is a key enzyme of the urea cycle that catalyses the conversion of arginine to ornithine and urea, which is the final cytosolic reaction of urea formation in the mammalian liver. The recombinant strain of the yeast Saccharomyces cerevisiae that is capable of overproducing arginase I (rhARG1) from human liver under the control of the efficient copper-inducible promoter CUP1, was constructed. The (His)(6)-tagged rhARG1 was purified in one step from the cell-free extract of the recombinant strain by metal-affinity chromatography with Ni-NTA agarose. The maximal specific activity of the 40-fold purified enzyme was 1600 µmol min(-1) mg(-1) protein.

Canavanine augments proapoptotic effects of arginine deprivation in cultured human cancer cells.

Arginine deprivation achieved by means of recombinant arginine-degrading enzymes is currently being developed as a novel anticancer enzymotherapy. In this study, we showed that arginine deprivation in vitro profoundly and selectively sensitized human cancer cells of different organ origin to low doses of canavanine, an arginine analogue of plant origin. In sensitive cancer cells arginine starvation led to the activation of caspase-9, caspase-3 and caspase-7, cleavage of reparation enzyme, polyADP ribosyl polymerase, and DNA fragmentation, which are the typical hallmarks of intrinsic apoptosis realized by the mitochondrial pathway. Co-administration of canavanine significantly accelerated and enhanced apoptotic manifestations induced by arginine deprivation. The augmentation of canavanine toxicity for cancer cells was observed when either a formulated arginine-free medium or complete medium supplemented with bovine arginase preparation was used. Cycloheximide efficiently rescued malignant cells from canavanine-induced cytotoxicity under arginine deprivation, suggesting that it results mainly from canavanine incorporation into newly synthesized proteins. Cancer cells sensitive or resistant to arginine deprivation alone were not capable of restoring their proliferation after 24 h of combined treatment, whereas pseudonormal cells retained such ability. Our data suggest that the incorporation of canavanine into anticancer treatment schemes based on artificially created arginine starvation could be a novel strategy in tumor enzymochemotherapy.

Cancer cell sensitivity to arginine deprivation in vitro is not determined by endogenous levels of arginine metabolic enzymes.

Single amino acid Arg (arginine) deprivation is currently considered as a therapeutic approach to treat certain types of tumours; the molecular mechanisms that underlie tumour cell sensitivity or resistance to Arg restriction are still little understood. Here, we address the question of whether endogenous levels of key Arg metabolic enzymes [catabolic: arginases, ARG1 (arginase type 1) and ARG2 (arginase type 2), and anabolic: OTC (ornithine transcarbamylase) and ASS (argininosuccinate synthetase)] affect cellular responses to arginine deprivation in vitro. Human epithelial cancer cells of different organs of origin exhibiting variable sensitivity to Arg deprivation provided the experimental models. Neither the basal expression status of the analysed enzymes, nor their changes upon arginine withdrawal correlated with cancer cell sensitivity to arginine deprivation. However, the ability to utilize exogenous Arg precursors (ornithine and citrulline) for growth in Arg-deficient medium strongly correlated with expression of the corresponding enzymes, OTC and ASS. We also observed that OTC expression was below the level of detection in all the types of tumour cells analysed, suggesting that in vitro, at least for them, Arg is an essential amino acid.

Combinatory Treatment of Canavanine and Arginine Deprivation Efficiently Targets Human Glioblastoma Cells via Pleiotropic Mechanisms.

Glioblastomas are the most frequent and aggressive form of primary brain tumors with no efficient cure. However, they often exhibit specific metabolic shifts that include deficiency in the biosynthesis of and dependence on certain exogenous amino acids. Here, we evaluated, in vitro, a novel combinatory antiglioblastoma approach based on arginine deprivation and canavanine, an arginine analogue of plant origin, using two human glioblastoma cell models, U251MG and U87MG. The combinatory treatment profoundly affected cell viability, morphology, motility and adhesion, destabilizing the cytoskeleton and mitochondrial network, and induced apoptotic cell death. Importantly, the effects were selective toward glioblastoma cells, as they were not pronounced for primary rat glial cells. At the molecular level, canavanine inhibited prosurvival kinases such as FAK, Akt and AMPK. Its effects on protein synthesis and stress response pathways were more complex and dependent on exposure time. We directly observed canavanine incorporation into nascent proteins by using quantitative proteomics. Although canavanine in the absence of arginine readily incorporated into polypeptides, no motif preference for such incorporation was observed. Our findings provide a strong rationale for further developing the proposed modality based on canavanine and arginine deprivation as a potential antiglioblastoma metabolic therapy independent of the blood-brain barrier.

Identification of hexose transporter-like sensor HXS1 and functional hexose transporter HXT1 in the methylotrophic yeast Hansenula polymorpha.

We identified in the methylotrophic yeast Hansenula polymorpha (syn. Pichia angusta) a novel hexose transporter homologue gene, HXS1 (hexose sensor), involved in transcriptional regulation in response to hexoses, and a regular hexose carrier gene, HXT1 (hexose transporter). The Hxs1 protein exhibits the highest degree of primary sequence similarity to the Saccharomyces cerevisiae transporter-like glucose sensors, Snf3 and Rgt2. When heterologously overexpressed in an S. cerevisiae hexose transporter-less mutant, Hxt1, but not Hxs1, restores growth on glucose or fructose, suggesting that Hxs1 is nonfunctional as a carrier. In its native host, HXS1 is expressed at moderately low level and is required for glucose induction of the H. polymorpha functional low-affinity glucose transporter Hxt1. Similarly to other yeast sensors, one conserved amino acid substitution in the Hxs1 sequence (R203K) converts the protein into a constitutively signaling form and the C-terminal region of Hxs1 is essential for its function in hexose sensing. Hxs1 is not required for glucose repression or catabolite inactivation that involves autophagic degradation of peroxisomes. However, HXS1 deficiency leads to significantly impaired transient transcriptional repression in response to fructose, probably due to the stronger defect in transport of this hexose in the hxs1Delta deletion strain. Our combined results suggest that in the Crabtree-negative yeast H. polymorpha, the single transporter-like sensor Hxs1 mediates signaling in the hexose induction pathway, whereas the rate of hexose uptake affects the strength of catabolite repression.

A Complex Scenario and Underestimated Challenge: The Tumor Microenvironment, ER Stress, and Cancer Treatment.

The paradoxical role of ER stress in malignant diseases is only just being unraveled and remains incompletely understood. A particular challenge is the complex interplay between spaciotemporal and locoregional microenvironmental constraints in solid tumors and stress responses upon treatment; thus, the potential for new combinatorial therapeutic options to foster the coincidence of ER stress-related deadly events is likely to be underestimated. Without claiming this review to be complete, we present a comprehensive overview of the signaling mechanisms associated with the unfolded protein response (UPR) and the molecular link to cell survival and death mechanisms. We (i) delineate the mechanistic scenario and outcome of the UPR; (ii) discuss the role of ER stress in cancer development and progression; (iii) highlight the impact of various environmental conditions and stress stimuli, such as nutrient limitation and tumor hypoxia, in this context; and (iv) attempt to shed some light on the putative link between DNA damage, irradiation, and ER stress to emphasize the potential of therapeutic targeting of ER stress pathways for combined cancer treatments.

Glucose regulation in the methylotrophic yeast Hansenula (Ogataea) polymorpha is mediated by a putative transceptor Gcr1.

The HpGcr1, a hexose transporter homologue from the methylotrophic yeast Hansenula (Ogataea) polymorpha, was previously identified as being involved in glucose repression. Intriguingly, potential HpGcr1 orthologues are found only in the genomes of a few yeasts phylogenetically closely related to H. polymorpha, but are absent in all other yeasts. The other closest HpGcr1 homologues are fungal high-affinity glucose symporters or putative transceptors suggesting a possible HpGcr1 origin due to a specific archaic gene retention or via horizontal gene transfer from Eurotiales fungi. Herein we report that, similarly to other yeast non-transporting glucose sensors, the substitution of the conserved arginine residue converts HpGcr1R165K into a constitutively signaling form. Synthesis of HpGcr1R165K in gcr1Δ did not restore glucose transport or repression but instead profoundly impaired growth independent of carbon source used. Simultaneously, gcr1Δ was impaired in transcriptional induction of repressible peroxisomal alcohol oxidase and in growth on methanol. Overexpression of the functional transporter HpHxt1 in gcr1Δ partially restored growth on glucose and glucose repression but did not rescue impaired growth on methanol. Heterologous expression of HpGcr1 in a Saccharomyces cerevisiae hxt-null strain did not restore glucose uptake due to protein mislocalization. However, HpGcr1 overexpression in H. polymorpha led to increased sensitivity to extracellular 2-deoxyglucose, suggesting HpGcr1 is a functional glucose carrier. The combined data suggest that HpGcr1 represents a novel type of yeast glucose transceptor functioning also in the absence of glucose.

Arginine Deprivation Therapy: Putative Strategy to Eradicate Glioblastoma Cells by Radiosensitization.

Tumor cells-even if nonauxotrophic-are often highly sensitive to arginine deficiency. We hypothesized that arginine deprivation therapy (ADT) if combined with irradiation could be a new treatment strategy for glioblastoma (GBM) patients because systemic ADT is independent of local penetration and diffusion limitations. A proof-of-principle in vitro study was performed with ADT being mimicked by application of recombinant human arginase or arginine-free diets. ADT inhibited two-dimensional (2-D) growth and cell-cycle progression, and reduced growth recovery after completion of treatment in four different GBM cell line models. Cells were less susceptible to ADT alone in the presence of citrulline and in a three-dimensional (3-D) environment. Migration and 3-D invasion were not unfavorably affected. However, ADT caused a significant radiosensitization that was more pronounced in a GBM cell model with p53 loss of function as compared with its p53-wildtype counterpart. The synergistic effect was independent of basic and induced argininosuccinate synthase or argininosuccinate lyase protein expression and not abrogated by the presence of citrulline. The radiosensitizing potential was maintained or even more distinguishable in a 3-D environment as verified in p53-knockdown and p53-wildtype U87-MG cells via a 60-day spheroid control probability assay. Although the underlying mechanism is still ambiguous, the observation of ADT-induced radiosensitization is of great clinical interest, in particular for patients with GBM showing high radioresistance and/or p53 loss of function. Mol Cancer Ther; 17(2); 393-406. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

Peroxisomes and peroxisomal transketolase and transaldolase enzymes are essential for xylose alcoholic fermentation by the methylotrophic thermotolerant yeast, Ogataea (Hansenula) polymorpha.

BACKGROUND: Ogataea (Hansenula) polymorpha is one of the most thermotolerant xylose-fermenting yeast species reported to date. Several metabolic engineering approaches have been successfully demonstrated to improve high-temperature alcoholic fermentation by O. polymorpha. Further improvement of ethanol production from xylose in O. polymorpha depends on the identification of bottlenecks in the xylose conversion pathway to ethanol. RESULTS: Involvement of peroxisomal enzymes in xylose metabolism has not been described to date. Here, we found that peroxisomal transketolase (known also as dihydroxyacetone synthase) and peroxisomal transaldolase (enzyme with unknown function) in the thermotolerant methylotrophic yeast, Ogataea (Hansenula) polymorpha, are required for xylose alcoholic fermentation, but not for growth on this pentose sugar. Mutants with knockout of DAS1 and TAL2 coding for peroxisomal transketolase and peroxisomal transaldolase, respectively, normally grow on xylose. However, these mutants were found to be unable to support ethanol production. The O. polymorpha mutant with the TAL1 knockout (coding for cytosolic transaldolase) normally grew on glucose and did not grow on xylose; this defect was rescued by overexpression of TAL2. The conditional mutant, pYNR1-TKL1, that expresses the cytosolic transketolase gene under control of the ammonium repressible nitrate reductase promoter did not grow on xylose and grew poorly on glucose media supplemented with ammonium. Overexpression of DAS1 only partially restored the defects displayed by the pYNR1-TKL1 mutant. The mutants defective in peroxisome biogenesis, pex3Δ and pex6Δ, showed normal growth on xylose, but were unable to ferment this sugar. Moreover, the pex3Δ mutant of the non-methylotrophic yeast, Scheffersomyces (Pichia) stipitis, normally grows on and ferments xylose. Separate overexpression or co-overexpression of DAS1 and TAL2 in the wild-type strain increased ethanol synthesis from xylose 2 to 4 times with no effect on the alcoholic fermentation of glucose. Overexpression of TKL1 and TAL1 also elevated ethanol production from xylose. Finally, co-overexpression of DAS1 and TAL2 in the best previously isolated O. polymorpha xylose to ethanol producer led to increase in ethanol accumulation up to 16.5 g/L at 45 °C; or 30-40 times more ethanol than is produced by the wild-type strain. CONCLUSIONS: Our results indicate the importance of the peroxisomal enzymes, transketolase (dihydroxyacetone synthase, Das1), and transaldolase (Tal2), in the xylose alcoholic fermentation of O. polymorpha.

A New Yeast Peroxin, Pex36, a Functional Homolog of Mammalian PEX16, Functions in the ER-to-Peroxisome Traffic of Peroxisomal Membrane Proteins.

Peroxisomal membrane proteins (PMPs) traffic to peroxisomes by two mechanisms: direct insertion from the cytosol into the peroxisomal membrane and indirect trafficking to peroxisomes via the endoplasmic reticulum (ER). In mammals and yeast, several PMPs traffic via the ER in a Pex3- and Pex19-dependent manner. In Komagataella phaffii (formerly called Pichia pastoris) specifically, the indirect traffic of Pex2, but not of Pex11 or Pex17, depends on Pex3, but all PMPs tested for indirect trafficking require Pex19. In mammals, the indirect traffic of PMPs also requires PEX16, a protein that is absent in most yeast species. In this study, we isolated PEX36, a new gene in K. phaffii, which encodes a PMP. Pex36 is required for cell growth in conditions that require peroxisomes for the metabolism of certain carbon sources. This growth defect in cells lacking Pex36 can be rescued by the expression of human PEX16, Saccharomyces cerevisiae Pex34, or by overexpression of the endogenous K. phaffii Pex25. Pex36 is not an essential protein for peroxisome proliferation, but in the absence of the functionally redundant protein, Pex25, it becomes essential and less than 20% of these cells show import-incompetent, peroxisome-like structures (peroxisome remnants). In the absence of both proteins, peroxisome biogenesis and the intra-ER sorting of Pex2 and Pex11C are seriously impaired, likely by affecting Pex3 and Pex19 function.