Translational landscape and protein biogenesis demands of the early secretory pathway in Komagataella phaffii

Abstract

Background: Eukaryotes use distinct networks of biogenesis factors to synthesize, fold, monitor, traffic, and secrete proteins. During heterologous expression, saturation of any of these networks may bottleneck titer and yield. To understand the flux through various routes into the early secretory pathway, we quantified the global and membrane-associated translatomes of Komagataella phaffii.

Results: By coupling Ribo-seq with long-read mRNA sequencing, we generated a new annotation of protein-encoding genes. By using Ribo-seq with subcellular fractionation, we quantified demands on co- and posttranslational translocation pathways. During exponential growth in rich media, protein components of the cell-wall represent the greatest number of nascent chains entering the ER. Transcripts encoding the transmembrane protein PMA1 sequester more ribosomes at the ER membrane than any others. Comparison to Saccharomyces cerevisiae reveals conservation in the resources allocated by gene ontology, but variation in the diversity of gene products entering the secretory pathway.

Conclusion: A subset of host proteins, particularly cell-wall components, impose the greatest biosynthetic demands in the early secretory pathway. These proteins are potential targets in strain engineering aimed at alleviating bottlenecks during heterologous protein production.

Keywords: Pichia pastoris; Protein secretion; Resource allocation; Ribosome profiling.

Fig. 1

Overview of Ribo-seq and subcellular fractionation. Ribosomes (grey) bound to a translocon (red) are only solubilized in the presence of detergent. The total sample has footprints originating from both membrane-bound and free-floating ribosomes. The soluble fraction is enriched in footprints from free-floating ribosomes. The membrane fraction is enriched in footprints from membrane-bound ribosomes

Fig. 2

Corrections applied to Ribo-seq data. a Ribosome-protected read counts at each codon were scaled by the total reads mapping to the ORF. Dots represent individual codons, and the line represents a composite of rolling means and medians see Methods. Regions in orange are the same width and are used to demonstrate that masked codons at the beginning of ORFs have a greater influence of calculated expression than masked codons at the end of ORFs. b Data from a after metagene correction. c Comparison of ribosome-protected reads per codon for highly expressed genes of different length. TPM for RPL5 gene is approximately 135% greater than TPM for YEF3 while producing approximately 38% as many ribosome-protected reads. After metagene correction cTPM scores are similar preserving the same difference in ribosome sequestration

Fig. 3

Protein expression and trafficking in K. phaffi. Tessellations are calculated using cTPM from the total fraction of a CHX treated culture and represent relative quantities of nascent chains produced from each gene. a Nascent chains produced by all ribosomes. b Nascent chains from genes showing twofold membrane enrichment. This includes mitochondrial and ER destined proteins. c Nascent chains from genes showing twofold membrane enrichment that are not predicted to be mitochondrial. d Nascent chains from genes showing less than twofold membrane enrichment but with a predicted ER signal sequence

Fig. 4

Comparison of translation from samples of membrane-bound and soluble fraction. Values are calculated using fractions obtained after incubation with CHX

Fig. 5

Nascent peptide length and membrane enrichment for secreted, lumenal, or GPI-anchored proteins. Proteins have a predicted N-terminal signal sequence. GPI anchors are included. The shaded box is drawn over genes with less than twofold membrane enrichment, which are considered posttranslationally targeted

Fig. 6

Correlation of membrane enrichment scores between species. Scores are deteremined using the membrane-bound and soluble fractions of ribosomes from cultures treated with CHX. a Enrichment scores restricted to signal sequence bearing proteins. Contrast dots represent genes found in Table 2. b Enrichment scores restricted to non-mitochondrial transmembrane proteins. c Enrichment scores restricted to mitochondrial proteins. d Enrichment scores restricted to cytosolic proteins

Fig. 7

Demands imposed on secretion pathway. Blue lines represent membrane proteins and orange lines represent secreted, lumenal or GPI-anchored proteins. a Demands in S. cerevisiae. b Demands in K. phaffii

Fig. 8

Demands imposed by different translocation pathways. a Cotranslational translocation of long protein and short proteins. b Translocation of short proteins which require both co- and posttranslational translocons c Posttranslational translocation