Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation - PubMed (original) (raw)

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

Ravi Manjithaya et al. J Cell Biol. 2010.

Abstract

In contrast to the enormous advances made regarding mechanisms of conventional protein secretion, mechanistic insights into the unconventional secretion of proteins are lacking. Acyl coenzyme A (CoA)-binding protein (ACBP; AcbA in Dictyostelium discoideum), an unconventionally secreted protein, is dependent on Golgi reassembly and stacking protein (GRASP) for its secretion. We discovered, surprisingly, that the secretion, processing, and function of an AcbA-derived peptide, SDF-2, are conserved between the yeast Pichia pastoris and D. discoideum. We show that in yeast, the secretion of SDF-2-like activity is GRASP dependent, triggered by nitrogen starvation, and requires autophagy proteins as well as medium-chain fatty acyl CoA generated by peroxisomes. Additionally, a phospholipase D implicated in soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor-mediated vesicle fusion at the plasma membrane is necessary, but neither peroxisome turnover nor fusion between autophagosomes and the vacuole is essential. Moreover, yeast Acb1 and several proteins required for its secretion are necessary for sporulation in P. pastoris. Our findings implicate currently unknown, evolutionarily conserved pathways in unconventional secretion.

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Figures

Figure 1.

Figure 1.

P. pastoris secretes Acb1 upon nitrogen starvation and processes it to generate SDF-2–like activity. (A) Cell-free supernatants were collected from either wild-type P. pastoris cells growing in rich medium or after 4 h in nitrogen starvation medium and tested for SDF-2–like activity using the bioassay as explained in Materials and methods. Samples were passed through an anion exchange chromatography, and the activity was eluted in 400 mM NaCl (salt eluate). An aliquot of this sample was protease treated (+ proteinase K; 20 µg) or incubated with either antibody to D. discoideum AcbA (1:500) or with antibody against GABA (1:5,000) and tested for SDF-2–like activity (Anjard and Loomis, 2005). Because KP cells can sporulate when presented with GABA, the experiment with antibody against GABA is a control to test whether the P. pastoris culture supernatant activated SDF-2 production in the KP cells by generating GABA. 10 nM recombinant P. pastoris HIS-Acb1 (PpAcb1) either untreated or digested with trypsin and purified (10 pM) were tested for SDF-2–like activity. (B) D. discoideum dhkA−/K (SDF-2 receptor null) cell bioassay. Induction was performed using 10 pM synthetic D. discoideum cytokinin and 1 pM SDF-2 peptides as controls. 1 µl supernatant from wild-type P. pastoris cells starved for 4 h was also tested. (C) Time course of the SDF-2–like activity secreted by P. pastoris cells. Samples were collected at the indicated times, purified, and quantified by the bioassay. (D) Acb1 is secreted and processed to generate SDF-2–like activity. Wild-type cells were nitrogen starved for 3 h before 1 µM TPCK was added to an aliquot of cells. 1 h later, the supernatant was collected from the untreated (none) and treated (+ TPCK) cells. TPCK was removed from part of the TPCK-treated sample and processed with trypsin as described in Materials and methods. All the samples were tested for SDF-2–like activity. Error bars indicate mean ± SD.

Figure 2.

Figure 2.

Primary sequence of P. pastoris Acb1 and activity of a P. pastoris protease that processes AcbA to SDF-2. (A) The primary sequences of Acbps from P. pastoris (NCBI Protein database accession no. XP_002490495), S. cerevisiae (NCBI Protein database accession no. NP_011551), Homo sapiens (NCBI Protein database accession no. NP_001073331), and D. discoideum (NCBI Protein database accession no. XP_646321) were aligned in CLUSTALW using the default alignment parameters (*, conserved residues). Putative, conserved trypsin-like cleavage site residues (K) are highlighted in boxed regions. The MatGAT program was used to calculate percent identity (I) and similarity (S) of the P. pastoris Acb1 protein with that of S. cerevisiae (I = 63.2%; S = 77%), H. sapiens (I = 55.2%; S = 77%), and D. discoideum (I = 45.3%; S = 62.8%). (B) Trypsin-like protease activity of acb1Δ cells. The acb1Δ cells were incubated in nitrogen starvation medium for 4 h without any addition (none) or in the presence of 100 pM recombinant AcbA, and extracellular media were collected, purified, and assayed for SDF-2–like activity (AcbA). 1 µM TPCK inhibitor was added 3 h after transfer to starvation medium and assayed (AcbA + TPCK). Part of the sample was digested with trypsin (see Materials and methods), purified, and assayed using the bioassay (AcbA + TPCK + trypsin). The GRASP-null mutant (grh1Δ) was also processed and tested for SDF-2–like activity like the other samples. Experiments were performed three times.

Figure 3.

Figure 3.

Autophagy and protein trafficking mutants affected in production of SDF-2–like activity. (A) Wild-type cells and various atg mutants were incubated in nitrogen starvation medium for 4 h, and cell supernatants were processed and tested for SDF-2–like activity. Cvt, cytosol to vacuole transport; MN, micronucleophagy. (B) Rapamycin induces Acb1 release. Wild-type (open circles) and atg1Δ cells (closed circles) were grown in SD+N medium up to 1 A600/ml, rapamycin (200 ng/ml final concentration) was added to the medium, and cell supernatants were collected at the indicated times, purified, and quantified by the bioassay. Experiments were performed three times.

Figure 4.

Figure 4.

Acb1 secretion in mutants affecting peroxisome biogenesis and metabolism. (A) Wild-type cells and various mutants were incubated in nitrogen starvation medium at 1 A600/ml for 4 h, and cell supernatants were tested for SDF-2–like activity. An aliquot of the wild-type cells was treated with 100 µg/ml cerulenin upon shift to starvation medium and assayed for SDF-2–like activity after 4 h. (B) Cell supernatants from pex3Δ, atg1Δ, grh1Δ, faa2Δ, and spo14Δ mutants starved for 4 h in nitrogen-deficient medium were treated with trypsin to see whether any secreted Acb1 could be processed to generate SDF-2–like activity. The wild-type controls that were analyzed in this experiment are from Fig. 1 D but are shown again for clarity. Experiments were performed three times.

Figure 5.

Figure 5.

Acb1 is necessary for sporulation. Wild-type, atg1Δ, atg8Δ, and acb1Δ cells were incubated in 1% potassium acetate solution at 30°C with rotation at 250 rpm. An aliquot of acb1Δ cells was incubated with 4 nM recombinant AcbA during the sporulation assay and analyzed for production of spores. 1 ml of the culture was processed as explained in Materials and methods to destroy viable cells. The spore preparation was appropriately diluted and plated on YPD plates. Plates were incubated for 2–3 d, and colonies were counted. Spore production for individual strains was expressed as the percentage of the wild-type spore count. One of two experiments is shown. Wild-type and acb1Δ cells were subjected to the sporulation assay as described in Materials and methods.

Figure 6.

Figure 6.

Working model for the unconventional secretion of Acb1 in P. pastoris. Pex11, a peroxisomal membrane protein associated with the peroxisome membrane, is transported to peroxisomes via the action of Pex3 and Pex19, whereas Faa2 is transported into peroxisomes via the PTS1 import pathway that relies on components of the peroxisomal importomer and the receptor-recycling machinery (see Results). Acb1 binds MCFA-CoA, which is derived by the Pex11-mediated transport of cytosolic MCFA into the peroxisome matrix, where the PTS1-containing protein Faa2 (fatty acyl CoA synthetase) couples it to CoA. The Acb1 bound to MCFA-CoA is captured in specialized autophagosomes (Acb1 vesicles) via the action of Atg11, involved in selective autophagy, and other core Atg proteins required for autophagosome formation. The normal fusion of Acb1 vesicles with vacuoles, but not that of general autophagic vesicles involved in macroautophagy, is bypassed during unconventional secretion. Acb1 vesicles might be delivered directly or indirectly for fusion with the plasma membrane, most likely via amphisomes. In addition to the proteins shown to be involved in Acb1 secretion, GRASP is also required. The fusion of a presently unknown vesicular compartment with the plasma membrane would release extracellular Acb1, which is then proteolytically cleaved by the P. pastoris trypsin-like protease, to generate extracellular SDF-2–like activity. We call this process the unconventional secretion via autophagosomes pathway.

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