Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting

Abstract

Background: Budding yeasts are often used to secrete foreign proteins, but the efficiency is variable. To identify roadblocks in the yeast secretory pathway, we used a monomeric superfolder GFP (msGFP) as a visual tracer in Saccharomyces cerevisiae and Pichia pastoris. Results: One roadblock for msGFP secretion is translocation into the ER. Foreign proteins are typically fused to the bipartite α-factor secretion signal, which consists of the signal sequence followed by the pro region. The α-factor signal sequence directs posttranslational translocation. For msGFP, posttranslational translocation is inefficient with the α-factor signal sequence alone but is stimulated by the pro region. This requirement for the pro region can be bypassed by using the Ost1 signal sequence, which has been shown to direct cotranslational translocation. A hybrid secretion signal consisting of the Ost1 signal sequence followed by the α-factor pro region drives efficient translocation followed by rapid ER export. A second roadblock for msGFP secretion in S. cerevisiae occurs during exit from the Golgi, when some of the msGFP molecules are diverted to the vacuole. Deletion of the sorting receptor Vps10 prevents vacuolar targeting of msGFP at the expense of missorting vacuolar hydrolases such as carboxypeptidase Y (CPY) to the culture medium. However, a truncation of Vps10 blocks vacuolar targeting of msGFP while permitting CPY to be sorted normally. Conclusions: With budding yeasts, if the secretion or processing of a foreign protein is poor, we recommend two options. First, use the Ost1 signal sequence to achieve efficient entry into the secretory pathway while avoiding the processing issues associated with the α-factor pro region. Second, truncate Vps10 to suppress diversion to the vacuole. These insights obtained with msGFP highlight the value of applying cell biological methods to study yeast secretion.

Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris

“…There have been previous reported instances where utilising the α-MF as a signal sequence has not resulted in the highest yield (Lin-Cereghino et al 2013; Ferrari et al 1997; Heiss et al 2015; Fitzgerald and Glick 2014). This phenomenon seems to be protein-dependent and different options are often examined during strain optimisation.…”

Cargo Capture and Bulk Flow in the Early Secretory Pathway

“…Examples include GFP, horseradish peroxidase, peroxisomal proteins, and bacterial beta-lactamase (Bard et al 2006, Brandizzi et al 2004, EidenPlach et al 2004, Hirz et al 2013, Shi et al 2007, Thor et al 2009, Wiedmann et al 1984. The lack of selectivity in secretion is often exploited to produce secreted biopharmaceuticals and industrial catalysts (De Meyer & Depicker 2014, Fitzgerald & Glick 2014, Loos et al 2011. CHO cells, fungi, and plants are usually employed.…”

Visualizing Secretory Cargo Transport in Budding Yeast

“…This problem can be alleviated by introducing the vps10-104 allele, which encodes a Vps10 protein that lacks one of the two sortilin homology domains (Jørgensen, Emr, & Winther, 1999). The Vps10-104 mutant receptor continues to recognize endogenous vacuolar proteases but no longer recognizes foreign secretory cargo proteins (Fitzgerald & Glick, 2014;Jørgensen et al, 1999). Our results also indicate that for unknown reasons, glycosylation of the secretory cargo further suppresses diversion to the vacuole (Casler et al, 2018).…”

“…Many soluble yeast secretory cargo proteins undergo post-translational translocation (Ast, Cohen, & Schuldiner, 2013;Ng, Brown, & Walter, 1996), but it was essential to direct DsRed-Express2-FKBP RD (C22V) for cotranslational translocation to ensure its passage across the ER membrane. We appended the Ost1 signal sequence, which efficiently directs cotranslational translocation (Fitzgerald & Glick, 2014). Addition of this signal sequence generates ER-localized DsRed-Express2-FKBP RD (C22V) tetramers that form aggregates (see the red channel in Video 1 and Fig.…”