Dataset Information

The membrane dynamics of pexophagy are influenced by Sar1p in Pichia pastoris.

ABSTRACT: Several Sec proteins including a guanosine diphosphate/guanosine triphosphate exchange factor for Sar1p have been implicated in autophagy. In this study, we investigated the role of Sar1p in pexophagy by expressing dominant-negative mutant forms of Sar1p in Pichia pastoris. When expressing sar1pT34N or sar1pH79G, starvation-induced autophagy, glucose-induced micropexophagy, and ethanol-induced macropexophagy are dramatically suppressed. These Sar1p mutants did not affect the initiation or expansion of the sequestering membranes nor the trafficking of Atg11p and Atg9p to these membranes during micropexophagy. However, the lipidation of Atg8p and assembly of the micropexophagic membrane apparatus, which are essential to complete the incorporation of the peroxisomes into the degradative vacuole, were inhibited when either Sar1p mutant protein was expressed. During macropexophagy, the expression of sar1pT34N inhibited the formation of the pexophagosome, whereas sar1pH79G suppressed the delivery of the peroxisome from the pexophagosome to the vacuole. The pexophagosome contained Atg8p in wild-type cells, but in cells expressing sar1pH79G these organelles contain both Atg8p and endoplasmic reticulum components as visualized by DsRFP-HDEL. Our results demonstrate key roles for Sar1p in both micro- and macropexophagy.

SUBMITTER: Schroder LA

PROVIDER: S-EPMC2575157 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The membrane dynamics of pexophagy are influenced by Sar1p in Pichia pastoris.

Schroder Laura A LA Ortiz Michael V MV Dunn William A WA

Molecular biology of the cell 20080903 11

Several Sec proteins including a guanosine diphosphate/guanosine triphosphate exchange factor for Sar1p have been implicated in autophagy. In this study, we investigated the role of Sar1p in pexophagy by expressing dominant-negative mutant forms of Sar1p in Pichia pastoris. When expressing sar1pT34N or sar1pH79G, starvation-induced autophagy, glucose-induced micropexophagy, and ethanol-induced macropexophagy are dramatically suppressed. These Sar1p mutants did not affect the initiation or expans ...[more]

PMID: 18768759

Similar Datasets

Project description:To take full advantage of recombinant Pichia pastoris (Komagataella phaffii) as a production system for heterologous proteins, the complex protein secretory process should be understood and optimised by circumventing bottlenecks. Typically, little or no attention has been paid to the fate of newly synthesised protein inside the cell, or its passage through the secretory pathway, and only the secreted product is measured. However, the system's productivity (i.e. specific production rate qp), includes productivity of secreted (qp,extra) plus intracellularly accumulated (qp,intra) protein. In bioreactor cultivations with P. pastoris producing penicillin G acylase, we studied the dynamics of product formation, i.e. both the specific product secretion (qp,extra) and product retention (qp,intra) as functions of time, as well as the kinetics, i.e. productivity in relation to specific growth rate (?). Within the time course, we distinguished (I) an initial phase with constant productivities, where the majority of product accumulated inside the cells, and qp,extra, which depended on ? in a bell-shaped manner; (II) a transition phase, in which intracellular product accumulation reached a maximum and productivities (intracellular, extracellular, overall) were changing; (III) a new phase with constant productivities, where secretion prevailed over intracellular accumulation, qp,extra was linearly related to ? and was up to three times higher than in initial phase (I), while qp,intra decreased 4-6-fold. We show that stress caused by heterologous protein production induces cellular imbalance leading to a secretory bottleneck that ultimately reaches equilibrium. This understanding may help to develop cultivation strategies for improving protein secretion from P. pastoris.Key Points• A novel concept for industrial bioprocess development.• A Relationship between biomass growth and product formation in P. pastoris.• A Three (3) phases of protein production/secretion controlled by the AOX1-promoter.• A Proof of concept in production of industrially relevant penicillin G acylase.

Project description:Over the past three decades, due to the universal application of Pichia yeast in the fermentation industry as well as the establishment of Pichia pastoris fermentation process for more than 30 years, the technology of the whole process has become very mature and has now reached a stagnated period of growth. However, studies and research conducted on the genomics of the classic fermentation process and the uncovering of the biological phenomena in the fermentation process from the point of view of high-throughput gene or protein is still in its early stages, and there is still insufficient data within this field. First, in collaboration with Agilent Company, we designed and prepared an expression microarray that could be used for the detection of Pichia pastoris transcriptomics. The transcriptomic changes in the five key technology steps (time points), during the fermentation process of Pichia pastoris would then be detected with the aid of an expression microarray. The five key steps of technology described above formed two important biological processes, namely, the limiting carbon source replacement and secondly, the fermentative production of exogenous proteins. The biological phenomena involved in these two processes were displayed and analyzed at the transcriptional level. In addition to this, with regard to the most important function in the fermentation process of Pichia pastoris, oxid-reduction, the metabolic drift process was analyzed and the important genes that might dominate the changes in the metabolic flux were discovered creatively by using the function tree method in this paper. This study was undertaken from the point of view of the transcriptome and the biological phenomena in the fermemntation process of Pichia pastoris. Both of which, were thoroughly explained during this study. The hope is for many more researchers to optimize the strain fermentation process, to produce proteins at the genetic level, as well as providing and obtaining new perspectives and detailed scientific data for the continued development within this field.

Dataset Information

The membrane dynamics of pexophagy are influenced by Sar1p in Pichia pastoris.

Publications

The membrane dynamics of pexophagy are influenced by Sar1p in Pichia pastoris.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets