Results
BioID can successfully tag proteins colocalized with
secreted
proteins
We first investigated if intracellular PPIs between each SecP and their
supporting SecMs can be measured using the BioID method. To do this,
each bait-BirA was expressed in HEK293 cells using the Flp-In™ system
(see materials and methods) for targeted integration of the transgenes
into the same genomic locus to ensure comparable transcription rates of
each transgene. Variations in mRNA level caused by random integration
can trigger adaptive response such as the unfolded protein response in
some cell lines which reciprocally alters the active PPIs network
involved in the secretion. We observed successful secretion of bait-BirA
proteins into culture supernatant, evaluated by Western blot (Fig. 2a).
Thus, the BirA fusion did not change the intracellular localization of
the model proteins, and it is expected that they enter the secretory
pathway where they are processed and packaged for secretion. We also
verified the biotinylation profile by western blot for each cell line in
the presence and absence of biotin. The biotinylation profile of the
bait-BirA cells is different when biotin is added to the culture with
substantial increased biotinylation of specific proteins, while no
obvious change is observed for WT (Fig. 2b), suggesting that BioID2
successfully tagged specific proteins within the cells. Colocalization
of the bait-BirA proteins and the biotinylated proteins was then studied
by multicolor co-immunofluorescence microscopy to test whether
biotinylated proteins are actual partners of the model proteins. The
results demonstrated successful labeling of the interactors by BirA
through colocalization of the biotin-labeled proteins and bait-BirA,
while WT did not show increased biotinylation under the same
experimental conditions (Fig. 3).
To quantify the colocalizations, we calculated different colocalization
metrics (see methods) from the images and compared to the WT (Table 1
and Fig. S1), and the results confirmed the specificity of the BioID
labeling system to tag the proximal proteins (closer Li’s ICQ value to
0.5 or Pearson’s R value to one demonstrates a dependent protein
staining pattern between the red and green channels).
WT cells revealed endogenous biotinylation
landscape
After successful tagging of the proximal proteins we aimed to identify
the interactions with each bait protein. For this, cells were lysed, and
biotinylated proteins were purified using streptavidin. Purified
proteins were digested by trypsin, and peptides were subjected to
LC-MS/MS, and the biotinylated proteins in samples were mapped using
MaxQuant (Tyanova, Temu, &
Cox, 2016). Differentially biotinylated proteins were then identified
in each sample compared to the WT using Perseus
(Tyanova & Cox, 2018). When
implemented in a WT control cell line, we identified proteins that are
biotinylated endogenously along with bona fide interactors. These
include proteins that bind biotin as a cofactor such as carboxylases
which is problematic with streptavidin-based protein detection
(Tytgat et al., 2015).
Although the extent to which the general endogenous biotinylation has
not been systematically quantified, the biotinylated proteins isolated
from the WT sample showed considerable overlap with interacting proteins
detected in other model protein samples, suggesting endogenous
biotinylation may be more pervasive than previously believed. Thus,
using the interaction partners detected from the WT sample as
background, we filtered out interactions detected in each model
bait-BirA sample that were likely a result of endogenous biotinylation.
Among the top differentially biotinylated proteins in bait-BirA samples,
the bait proteins showed the highest log-fold change (LFC) (Fig. 4 and
TableS1). This observation is expected because the bait protein is a
potential substrate for BirA located in the closest vicinity of the
enzyme and is considered as evidence to show the biotinylation system is
working properly within the cell.
Interactors are enriched for secretory pathway components
and co-secreted
proteins
For each secreted protein we identified probable PPIs as interactors
having 3-fold or greater enrichment and an adjusted p-value <
0.1, in model proteins compared to WT control (Fig. S2 for all
thresholds and Fig. 5b for all significant interactions) based on
secretory pathway-related gold standards compiled from 3 independent
gene sets (see methods). We saw a significant enrichment for the
secretory pathway machinery, secretory-resident and co-secreted proteins
among probable PPIs across all model protein samples (Fig. 5a and Fig.
S3). The secretory machinery components are more enriched among the top
300 hits for all model proteins than other co-secreted proteins,
suggesting more frequent interactions between the secretory pathway
machinery and their products than the crosstalk between co-secreted
proteins. Probable PPIs detected in all model proteins (n=19) and hits
shared among all SERPIN gene products are significantly enriched for
proteins involved in protein folding (Fig. 5b). Indeed, molecular
chaperones are highly promiscuous when assisting protein folding due to
their inherent flexibility
(Mayer, 2010). Apart from
the shared interactions, PPIs for each model protein differ
substantially. Thus, the question remained if these private interactions
correspond to unique properties of each model protein.
Private interactors reflect post-translational and
structural features of model
proteins
PPIs in secretory pathway mediate the folding, modification and
transportation of secreted proteins
(Bonifacino
& Glick, 2004; Calakos, Bennett, Peterson, & Scheller, 1994; Ikawa et
al., 1997; Pearl & Prodromou, 2006; Watanabe et al., 2019).
Incidentally, co-expression analysis has linked certain PTMs across the
secretome to the expression of their responsible enzymes. For example,
PDIs are consistently upregulated in tissues secreting disulfide-rich
proteins (Feizi et al.,
2017). As the bait-BirA proteins differ in structural composition and
PTMs (Fig. 6), we wondered if bait-BirA proteins with shared features
have higher affinity for specific interactors. More specifically, we
hypothesize that proteins requiring a specific PTM would preferentially
interact with the secretory machinery components responsible for the PTM
synthesis. To establish a more comprehensive connection around
product-specific interactions with the secretory pathway, we aggregated
various PTM and structural properties across all model proteins and
analyzed their associations with the corresponding secretory machinery
using a Bayesian modeling framework (see methods). Among the studied
PTMs, bait-BirA proteins with disulfide bonds and N-linked glycans
demonstrated higher affinity towards specific interactions (fig. 7a)
that are known to help secretion of proteins with the corresponding
PTMs. Thus, we analyzed the detected interactions associated with
glycosylation, disulfide bond addition, and protein folding.
Proteins with increased glycosylation are associated with
quality control pathways
We detected significant interactions in the Calnexin/Calreticulin cycle
and related processes for more heavily glycosylated proteins (Fig. 7a).
For example, the glycosylated baits interacted with calreticulin (CALR),
a calcium-binding chaperone that promotes folding, oligomeric assembly,
and quality control of glycoproteins in the ER
(Nauseef, McCormick, &
Clark, 1995). They also interacted with UGGT1, which recognizes
glycoproteins with minor folding defects and reglucosylates single
N-glycans near the misfolded part of the protein. Reglucosylated
proteins are then recognized by CALR for recycling to the ER and
refolding or degradation
(Ferris, Jaber, Molinari,
Arvan, & Kaufman, 2013). Two members of the PDI family, PDIA3 and
ERp29, which form a complex with calreticulin/calnexin, also showed
association with N-glycosylated baites (Fig. 7a) suggesting their role
in glycoprotein folding and quality control. Calnexin/Calreticulin-PDIA3
complexes promote the oxidative folding of nascent polypeptides
(Sakono, Seko, Takeda, &
Ito, 2014) and ERp29 promotes isomerization of peptidyl-prolyl bonds to
attain the native polypeptide structure
(Sakono et al., 2014;
Tannous, Pisoni, Hebert, & Molinari, 2015). Proteins with chaperone
activity, such as HSPA5 (Fig. 7a), were also found to interact with
N-linked glycan-containing bait-BirA. HSPA5 is a component of the
glycoprotein quality-control (GQC) system which recognizes glycoproteins
with amino acid substitutions, and targets them for ER‐associated
degradation (ERAD) (Ferris,
Kodali, & Kaufman, 2014). EDEM3, another interactor associated with
the N-glycan containing proteins (Fig. 7a), is a glycosyltransferase
involved in ERAD mediated degradation of glycoproteins by catalyzing
mannose trimming from Man8GlcNAc2 to Man7GlcNAc2 in N-glycans
(Ninagawa et al., 2014).
Given that most of these molecular chaperones and enzymes are involved
in ERAD mediated degradation of the misfolded glycoproteins these
findings suggest the quality control pathways are critical for
synthesizing and secreting proteins with N-linked glycans.
Disulfide bond formation is rate-limiting in protein
secretion
Several members of the PDI family including P4HB, PDIA3, PDIA4 and PDIA6
significantly interacted with model-BirAs containing more disulfide
bonds (Fig. 7a). These enzymes catalyze the formation, breakage and
rearrangement of disulfide bonds through the thioredoxin-like domains
(Kozlov, Määttänen, Thomas,
& Gehring, 2010). The identification of various PDIs highlights the
importance of the oxidative folding enzymes in protein folding and
maintaining stability that can limit the efficiency of protein
secretion. The proteins with more disulfide bonds also interact with
major ER chaperones HSAP5 and DNAJB11, a co-chaperone of HSAP5, that
play a key role in protein folding and quality control in the ER lumen
(Ng, Watowich, & Lamb,
1992; Yu, Haslam, & Haslam, 2000), highlighting their important role
in secretion of the disulfide bond enriched proteins. The PDI, ERp44
showed the strongest association (LFC > 8, Fig. S4) with
disulfide bond enriched proteins i.e. SERPINC1 and SERPING1. ERp44
mediates the ER localization of the oxidoreductase Ero1α (an
oxidoreductin that reoxidizes P4HB to enable additional rounds of
disulfide formation) through the formation of reversible mixed
disulfides (Anelli et al.,
2003). Hence, the strong association of ERp44 suggests the importance
of the thiol-mediated retention in disulfide bond formation,
particularly when secretory is loaded with the proteins with dominant
disulfide bond. In addition, ERO1LB, PRDX4 and SIL1 were ER-localized
enzymes that were associated with disulfide bond formation. ERO1LB
efficiently reoxidizes P4HB
(Mezghrani et al., 2001),
PRDX4 couples hydrogen peroxide catabolism with oxidative protein
folding by reducing hydrogen peroxide
(Zito, 2013), and SIL1 can
reverse HSAP5 cysteine oxidation which alters its chaperone activity to
cope with suboptimal folding conditions
(Siegenthaler, Pareja, Wang,
& Sevier, 2017). The identification of these oxidoreductase enzymes
highlights the importance of ER redox homeostasis in disulfide bond
formation and protecting cells from the consequences of misfolded
proteins.
To validate the importance of the ERp44 interaction, which showed the
highest fold change, on productivity of proteins with more disulfide
bonds we knocked down ERP44 in the cells expressing SERPINC1-BirA, using
an orthogonal RNAi approach, esiRNAs, to target gene knockdown
(Kittler, Heninger, Franke,
Habermann, & Buchholz, 2005). HEK293 cells expressing SERPINC1-BirA
were transfected with esiRNA against ERP44, with EGFP and KIF11 as
negative/positive controls, respectively. The supernatants of esiRNA
experiments collected 48 and 72 h post-transfection for measuring the
secretion of SERPINC1-BirA by ELISA. All quantifications were repeated
in duplicate. The results showed ERP44 knockdown in SERPINC1-BirA cells
led to the 44% and 41% reduction in the secretion of SERPINC1 at day
two and three post-transfection, respectively, supporting the importance
of the Thiol-mediated retention on secretion of the disulfide bond
enriched protein.
Identified PPIs are associated with structural motifs on
bait
proteins
In addition to PTMs, the Bayesian modeling framework found associations
between SecP structural features and the SecMs (Fig. 7b). For example,
model proteins depleted in the asx motif
(Golovin & Henrick, 2008)
showed higher tendency to interact with DNAJB1, a molecular chaperone of
the HSP70 family. The asx motif impacts N-glycan occupancy of
Asn-X-Thr/Ser sites, depending on the ability of the peptide to adopt an
Asx-turn motif
(Imperiali, Shannon, &
Rickert, 1992; Imperiali, Shannon, Unno, & Rickert, 1992). As another
example, NUCB1, a chaperone-like amyloid binding protein that prevents
protein aggregation
(Bonito-Oliva, Barbash,
Sakmar, & Graham, 2017), interacted more strongly with our proteins
with more ST-turns (Fig. 7b). ST turns occur frequently at the N-termini
of α-helices (Doig, Stapley,
Macarthur, & Thornton, 2008) and are regarded as helix capping
features which stabilize α-helices in proteins
(Aurora & Rosee, 1998).
Thus, the enriched interaction of the NUCB1 with St-turn suggests that
it can help stabilize folding of protein with a predominant α-helical
secondary structure.