Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.

Depending on endoplasmic reticulum (ER) stress levels, the ER transmembrane multidomain protein IRE1α promotes either adaptation or apoptosis. Unfolded ER proteins cause IRE1α lumenal domain homo-oligomerization, inducing trans autophosphorylation that further drives homo-oligomerization of its cytosolic kinase/endoribonuclease (RNase) domains to activate mRNA splicing of adaptive XBP1 transcription factor. However, under high/chronic ER stress, IRE1α surpasses an oligomerization threshold that expands RNase substrate repertoire to many ER-localized mRNAs, leading to apoptosis. To modulate these effects, we developed ATP-competitive IRE1α Kinase-Inhibiting RNase Attenuators-KIRAs-that allosterically inhibit IRE1α's RNase by breaking oligomers. One optimized KIRA, KIRA6, inhibits IRE1α in vivo and promotes cell survival under ER stress. Intravitreally, KIRA6 preserves photoreceptor functional viability in rat models of ER stress-induced retinal degeneration. Systemically, KIRA6 preserves pancreatic ß cells, increases insulin, and reduces hyperglycemia in Akita diabetic mice. Thus, IRE1α powerfully controls cell fate but can itself be controlled with small molecules to reduce cell degeneration.

Trans-endocytosis of intact IL-15Rα-IL-15 complex from presenting cells into NK cells favors signaling for proliferation.

Interleukin 15 (IL-15) is an essential cytokine for the survival and proliferation of natural killer (NK) cells. IL-15 activates signaling by the ß and common γ (γc) chain heterodimer of the IL-2 receptor through trans-presentation by cells expressing IL-15 bound to the α chain of the IL-15 receptor (IL-15Rα). We show here that membrane-associated IL-15Rα-IL-15 complexes are transferred from presenting cells to NK cells through trans-endocytosis and contribute to the phosphorylation of ribosomal protein S6 and NK cell proliferation. NK cell interaction with soluble or surface-bound IL-15Rα-IL-15 complex resulted in Stat5 phosphorylation and NK cell survival at a concentration or density of the complex much lower than required to stimulate S6 phosphorylation. Despite this efficient response, Stat5 phosphorylation was reduced after inhibition of metalloprotease-induced IL-15Rα-IL-15 shedding from trans-presenting cells, whereas S6 phosphorylation was unaffected. Conversely, inhibition of trans-endocytosis by silencing of the small GTPase TC21 or expression of a dominant-negative TC21 reduced S6 phosphorylation but not Stat5 phosphorylation. Thus, trans-endocytosis of membrane-associated IL-15Rα-IL-15 provides a mode of regulating NK cells that is not afforded to IL-2 and is distinct from activation by soluble IL-15. These results may explain the strict IL-15 dependence of NK cells and illustrate how the cellular compartment in which receptor-ligand interaction occurs can influence functional outcome.

Size-dependent secretory protein reflux into the cytosol in association with acute endoplasmic reticulum stress.

Once secretory proteins have been targeted to the endoplasmic reticulum (ER) lumen, the proteins typically remain partitioned from the cytosol. If the secretory proteins misfold, they can be unfolded and retrotranslocated into the cytosol for destruction by the proteasome by ER-Associated protein Degradation (ERAD). Here, we report that correctly folded and targeted luminal ER fluorescent protein reporters accumulate in the cytosol during acute misfolded secretory protein stress in yeast. Photoactivation fluorescence microscopy experiments reveal that luminal reporters already localized to the ER relocalize to the cytosol, even in the absence of essential ERAD machinery. We named this process "ER reflux." Reflux appears to be regulated in a size-dependent manner for reporters. Interestingly, prior heat shock stress also prevents ER stress-induced reflux. Together, our findings establish a new ER stress-regulated pathway for relocalization of small luminal secretory proteins into the cytosol, distinct from the ERAD and preemptive quality control pathways. Importantly, our results highlight the value of fully characterizing the cell biology of reporters and describe a simple modification to maintain luminal ER reporters in the ER during acute ER stress.

The Development and Enhancement of FRAP as a Key Tool for Investigating Protein Dynamics.

The saga of fluorescence recovery after photobleaching (FRAP) illustrates how disparate technical developments impact science. Starting with the classic 1976 Axelrod et al. work in Biophysical Journal, FRAP (originally fluorescence photobleaching recovery) opened the door to extraction of quantitative information from photobleaching experiments, laying the experimental and theoretical groundwork for quantifying both the mobility and the mobile fraction of a labeled population of proteins. Over the ensuing years, FRAP's reach dramatically expanded, with new developments in GFP technology and turn-key confocal microscopy, which enabled measurement of protein diffusion and binding/dissociation rates in virtually every compartment within the cell. The FRAP technique and data catalyzed an exchange of ideas between biophysicists studying membrane dynamics, cell biologists focused on intracellular dynamics, and systems biologists modeling the dynamics of cell activity. The outcome transformed the field of cellular biology, leading to a fundamental rethinking of long-held theories of cellular dynamism. Here, we review the pivotal FRAP studies that made these developments and conceptual changes possible, which gave rise to current models of complex cell dynamics.

Connexin Type and Fluorescent Protein Fusion Tag Determine Structural Stability of Gap Junction Plaques.

Gap junctions (GJs) are made up of plaques of laterally clustered intercellular channels and the membranes in which the channels are embedded. Arrangement of channels within a plaque determines subcellular distribution of connexin binding partners and sites of intercellular signaling. Here, we report the discovery that some connexin types form plaque structures with strikingly different degrees of fluidity in the arrangement of the GJ channel subcomponents of the GJ plaque. We uncovered this property of GJs by applying fluorescence recovery after photobleaching to GJs formed from connexins fused with fluorescent protein tags. We found that connexin 26 (Cx26) and Cx30 GJs readily diffuse within the plaque structures, whereas Cx43 GJs remain persistently immobile for more than 2 min after bleaching. The cytoplasmic C terminus of Cx43 was required for stability of Cx43 plaque arrangement. We provide evidence that these qualitative differences in GJ arrangement stability reflect endogenous characteristics, with the caveat that the sizes of the GJs examined were necessarily large for these measurements. We also uncovered an unrecognized effect of non-monomerized fluorescent protein on the dynamically arranged GJs and the organization of plaques composed of multiple connexin types. Together, these findings redefine our understanding of the GJ plaque structure and should be considered in future studies using fluorescent protein tags to probe dynamics of highly ordered protein complexes.

Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat.

Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly.

Going Viral with Fluorescent Proteins.

Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses.

An in vitro compartmentalization-based method for the selection of bond-forming enzymes from large libraries.

We have developed a generalized in vitro compartmentalization-based bead display selection strategy that allows for the identification of enzymes that can perform ligation reactions. Although a number of methods have been developed to evolve such enzymes, many of them are limited in library size (10(6) -10(7) ), do not select for enzymes using a scheme that allows for multiple turnover, or only work on enzymes specific to nucleic acids. This approach is amenable to screening libraries of up to 10(12) protein variants by allowing beads to be overloaded with up to 10(4) unique mutants. Using this approach we isolated a variant of sortase A from Staphylococcus aureus that shows a 114-fold enhancement in kcat /KM in the absence of calcium compared to the wild-type and improved resistance to the inhibitory effects of cell lysates. Unlike the wild-type protein, the newly selected variant shows intracellular activity in the cytoplasm of eukaryotic cells where it may prove useful for intracellular labeling or synthetic biological applications. Biotechnol. Bioeng. 2016;113: 1647-1657. © 2016 Wiley Periodicals, Inc.

Static retention of the lumenal monotopic membrane protein torsinA in the endoplasmic reticulum.

TorsinA is a membrane-associated enzyme in the endoplasmic reticulum (ER) lumen that is mutated in DYT1 dystonia. How it remains in the ER has been unclear. We report that a hydrophobic N-terminal domain (NTD) directs static retention of torsinA within the ER by excluding it from ER exit sites, as has been previously reported for short transmembrane domains (TMDs). We show that despite the NTD's physicochemical similarity to TMDs, it does not traverse the membrane, defining torsinA as a lumenal monotopic membrane protein and requiring a new paradigm to explain retention. ER retention and membrane association are perturbed by a subset of nonconservative mutations to the NTD, suggesting that a helical structure with defined orientation in the membrane is required. TorsinA preferentially enriches in ER sheets, as might be expected for a lumenal monotopic membrane protein. We propose that the principle of membrane-based protein sorting extends to monotopic membrane proteins, and identify other proteins including the monotopic lumenal enzyme cyclooxygenase 1 (prostaglandin H synthase 1) that share this mechanism of retention with torsinA.

ERdj3 regulates BiP occupancy in living cells.

Co-chaperones regulate chaperone activities and are likely to impact a protein-folding environment as much as the chaperone itself. As co-chaperones are expressed substoichiometrically, the ability of co-chaperones to encounter a chaperone is crucial for chaperone activity. ERdj3, an abundant soluble endoplasmic reticulum (ER) co-chaperone of the Hsp70 BiP, stimulates the ATPase activity of BiP to increase BiP's affinity for client (or substrate) proteins. We investigated ERdj3 availability, how ERdj3 levels impact BiP availability, and the significance of J proteins for regulating BiP binding of clients in living cells. FRAP analysis revealed that overexpressed ERdj3-sfGFP dramatically decreases BiP-GFP mobility in a client-dependent manner. By contrast, ERdj3-GFP mobility remains low regardless of client protein levels. Native gels and co-immunoprecipitations established that ERdj3 associates with a large complex including Sec61α. Translocon binding probably ensures rapid encounters between emerging nascent peptides and stimulates BiP activity in the crucial early stages of secretory protein folding. Importantly, mutant BiP exhibited significantly increased mobility when it could not interact with any ERdjs. Thus, ERdjs appear to play the dual roles of increasing BiP affinity for clients and regulating delivery of clients to BiP. Our data suggest that BiP engagement of clients is enhanced in ER subdomains enriched in ERdj proteins.

Imaging the alphavirus exit pathway.

UNLABELLED: Alphaviruses are small enveloped RNA viruses with highly organized structures that exclude host cell proteins. They contain an internal nucleocapsid and an external lattice of the viral E2 and E1 transmembrane proteins. Alphaviruses bud from the plasma membrane (PM), but the process and dynamics of alphavirus assembly and budding are poorly understood. Here we generated Sindbis viruses (SINVs) with fluorescent protein labels on the E2 envelope protein and exploited them to characterize virus assembly and budding in living cells. During virus infection, E2 became enriched in localized patches on the PM and in filopodium-like extensions. These E2-labeled patches and extensions contained all of the viral structural proteins. Correlative light and electron microscopy studies established that the patches and extensions colocalized with virus budding structures, while light microscopy showed that they excluded a freely diffusing PM marker protein. Exclusion required the interaction of the E2 protein with the capsid protein, a critical step in virus budding, and was associated with the immobilization of the envelope proteins on the cell surface. Virus infection induced two distinct types of extensions: tubulin-negative extensions that were ∼2 to 4 µm in length and excluded the PM marker, and tubulin-positive extensions that were >10 µm long, contained the PM marker, and could transfer virus particles to noninfected cells. Tubulin-positive extensions were selectively reduced in cells infected with a nonbudding SINV mutant. Together, our data support a model in which alphavirus infection induces reorganization of the PM and cytoskeleton, leading to virus budding from specialized sites. IMPORTANCE: Alphaviruses are important and widely distributed human pathogens for which vaccines and antiviral therapies are urgently needed. These small highly organized viruses bud from the host cell PM. Virus assembly and budding are critical but little understood steps in the alphavirus life cycle. We developed alphaviruses with fluorescent protein tags on one of the viral membrane (envelope) proteins and used a variety of microscopy techniques to follow the envelope protein and a host cell PM protein during budding. We showed that alphavirus infection induced the formation of patches and extensions on the PM where the envelope proteins accumulate. These sites excluded other PM proteins and correlated with virus budding structures. Exclusion of PM proteins required specific interactions of the viral envelope proteins with the internal capsid protein. Together, our data indicate that alphaviruses extensively reorganize the cell surface and cytoskeleton to promote their assembly and budding.

Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions.

Several fluorescent proteins (FPs) are prone to forming low-affinity oligomers. This undesirable tendency is exacerbated when FPs are confined to membranes or when fused to naturally oligomeric proteins. Oligomerization of FPs limits their suitability for creating fusions with proteins of interest. Unfortunately, no standardized method evaluates the biologically relevant oligomeric state of FPs. Here, we describe a quantitative visual assay for assessing whether FPs are sufficiently monomeric under physiologic conditions. Membrane-associated FP-fusion proteins, by virtue of their constrained planar geometry, achieve high effective concentrations. We exploited this propensity to develop an assay to measure FP tendencies to oligomerize in cells. FPs were fused on the cytoplasmic end of an endoplasmic reticulum (ER) signal-anchor membrane protein (CytERM) and expressed in cells. Cells were scored based on the ability of CytERM to homo-oligomerize with proteins on apposing membranes and restructure the ER from a tubular network into organized smooth ER (OSER) whorl structures. The ratio of nuclear envelope and OSER structures mean fluorescent intensities for cells expressing enhanced green fluorescent protein (EGFP) or monomeric green fluorescent protein (mGFP) CytERM established standards for comparison of uncharacterized FPs. We tested three FPs and identified two as sufficiently monomeric, while a third previously reported as monomeric was found to strongly oligomerize.

Mechanism of collapse of endoplasmic reticulum cisternae during African swine fever virus infection.

Infection of cells with African swine fever virus (ASFV) can lead to the formation of zipper-like stacks of structural proteins attached to collapsed endoplasmic reticulum (ER) cisternae. We show that the collapse of ER cisternae observed during ASFV infection is dependent on the viral envelope protein, J13Lp. Expression of J13Lp alone in cells is sufficient to induce collapsed ER cisternae. Collapse was dependent on a cysteine residue in the N-terminal domain of J13Lp exposed to the ER lumen. Luminal collapse was also dependent on the expression of J13Lp within stacks of ER where antiparallel interactions between the cytoplasmic domains of J13Lp orientated N-terminal domains across ER cisternae. Cisternal collapse was then driven by disulphide bonds between N-terminal domains arranged in antiparallel arrays across the ER lumen. This provides a novel mechanism for biogenesis of modified stacks of ER present in cells infected with ASFV, and may also be relevant to cellular processes.

Biochemical and cellular analysis of human variants of the DYT1 dystonia protein, TorsinA/TOR1A.

Early-onset dystonia is associated with the deletion of one of a pair of glutamic acid residues (c.904_906delGAG/c.907_909delGAG; p.Glu302del/Glu303del; ΔE 302/303) near the carboxyl-terminus of torsinA, a member of the AAA(+) protein family that localizes to the endoplasmic reticulum lumen and nuclear envelope. This deletion commonly underlies early-onset DYT1 dystonia. While the role of the disease-causing mutation, torsinAΔE, has been established through genetic association studies, it is much less clear whether other rare human variants of torsinA are pathogenic. Two missense variations have been described in single patients: R288Q (c.863G>A; p.Arg288Gln; R288Q) identified in a patient with onset of severe generalized dystonia and myoclonus since infancy and F205I (c.613T>A, p.Phe205Ile; F205I) in a psychiatric patient with late-onset focal dystonia. In this study, we have undertaken a series of analyses comparing the biochemical and cellular effects of these rare variants to torsinAΔE and wild-type (wt) torsinA to reveal whether there are common dysfunctional features. The results revealed that the variants, R288Q and F205I, are more similar in their properties to torsinAΔE protein than to torsinAwt. These findings provide functional evidence for the potential pathogenic nature of these rare sequence variants in the TOR1A gene, thus implicating these pathologies in the development of dystonia.

Proinsulin intermolecular interactions during secretory trafficking in pancreatic ß cells.

Classically, exit from the endoplasmic reticulum (ER) is rate-limiting for secretory protein trafficking because protein folding/assembly occurs there. In this study, we have exploited "hPro-CpepSfGFP," a human proinsulin bearing "superfolder" green fluorescent C-peptide expressed in pancreatic ß cells where it is processed to human insulin and CpepSfGFP. Remarkably, steady-state accumulation of hPro-CpepSfGFP and endogenous proinsulin is in the Golgi region, as if final stages of protein folding/assembly were occurring there. The Golgi regional distribution of proinsulin is dynamic, influenced by fasting/refeeding, and increased with ß cell zinc deficiency. However, coexpression of ER-entrapped mutant proinsulin-C(A7)Y shifts the steady-state distribution of wild-type proinsulin to the ER. Endogenous proinsulin coprecipitates with hPro-CpepSfGFP and even more so with hProC(A7)Y-CpepSfGFP. Using Cerulean and Venus-tagged proinsulins, we find that both WT-WT and WT-mutant proinsulin pairs exhibit FRET. The data demonstrate that wild-type proinsulin dimerizes within the ER but accumulates at a poorly recognized slow step within the Golgi region, reflecting either slow kinetics of proinsulin hexamerization, steps in formation of nascent secretory granules, or other unknown molecular events. However, in the presence of ongoing misfolding of a subpopulation of proinsulin in ß cells, the rate-limiting step in transport of the remaining proinsulin shifts to the ER.

Endoplasmic reticulum polymers impair luminal protein mobility and sensitize to cellular stress in alpha1-antitrypsin deficiency.

UNLABELLED: Point mutants of alpha1 -antitrypsin (α1AT) form ordered polymers that are retained as inclusions within the endoplasmic reticulum (ER) of hepatocytes in association with neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. These inclusions cause cell damage and predispose to ER stress in the absence of the classical unfolded protein response (UPR). The pathophysiology underlying this ER stress was explored by generating cell models that conditionally express wild-type (WT) α1AT, two mutants that cause polymer-mediated inclusions and liver disease (E342K [the Z allele] and H334D) and a truncated mutant (Null Hong Kong; NHK) that induces classical ER stress and is removed by ER-associated degradation. Expression of the polymeric mutants resulted in gross changes in the ER luminal environment that recapitulated the changes observed in liver sections from individuals with PI*ZZ α1AT deficiency. In contrast, expression of NHK α1AT caused electron lucent dilatation and expansion of the ER throughout the cell. Photobleaching microscopy in live cells demonstrated a decrease in the mobility of soluble luminal proteins in cells that express E342K and H334D α1AT, when compared to those that express WT and NHK α1AT (0.34 ± 0.05, 0.22 ± 0.03, 2.83 ± 0.30, and 2.84 ± 0.55 µm(2) /s, respectively). There was no effect on protein mobility within ER membranes, indicating that cisternal connectivity was not disrupted. Polymer expression alone was insufficient to induce the UPR, but the resulting protein overload rendered cells hypersensitive to ER stress induced by either tunicamycin or glucose depletion. CONCLUSION: Changes in protein diffusion provide an explanation for the cellular consequences of ER protein overload in mutants that cause inclusion body formation and α1AT deficiency.

Superfolder GFP is fluorescent in oxidizing environments when targeted via the Sec translocon.

The ability to study proteins in live cells using genetically encoded fluorescent proteins (FPs) has revolutionized cell biology (1-3). Researchers have created numerous FP biosensors and optimized FPs for specific organisms and subcellular environments in a rainbow of colors (4,5). However, expressing FPs in oxidizing environments such as the eukaryotic endoplasmic reticulum (ER) or the bacterial periplasm can impair folding, thereby preventing fluorescence (6,7). A substantial fraction of enhanced green fluorescent protein (EGFP) oligomerizes to form non-fluorescent mixed disulfides in the ER (6) and EGFP does not fluoresce in the periplasm when targeted via the SecYEG translocon (7). To overcome these obstacles, we exploited the highly efficient folding capability of superfolder GFP (sfGFP) (8). Here, we report sfGFP does not form disulfide-linked oligomers in the ER and maltose-binding protein (MBP) signal sequence (peri)-sfGFP (9) is brightly fluorescent in the periplasm of Escherichia coli. Thus, sfGFP represents an important research tool for studying resident proteins of oxidizing environments.

ERdj4 protein is a soluble endoplasmic reticulum (ER) DnaJ family protein that interacts with ER-associated degradation machinery.

Protein localization within cells regulates accessibility for interactions with co-factors and substrates. The endoplasmic reticulum (ER) BiP co-factor ERdj4 is up-regulated by ER stress and has been implicated in ER-associated degradation (ERAD) of multiple unfolded secretory proteins. Several other ERdj family members tend to interact selectively with nascent proteins, presumably because those ERdj proteins associate with the Sec61 translocon that facilitates entry of nascent proteins into the ER. How ERdj4 selects and targets terminally misfolded proteins for destruction remains poorly understood. In this study, we determined properties of ERdj4 that might aid in this function. ERdj4 was reported to retain its signal sequence and to be resistant to mild detergent extraction, suggesting that it was an integral membrane protein. However, live cell photobleaching analyses of GFP-tagged ERdj4 revealed that the protein exhibits diffusion coefficients uncommonly high for an ER integral membrane protein and more similar to the mobility of a soluble luminal protein. Biochemical characterization established that the ERdj4 signal sequence is cleaved to yield a soluble protein. Importantly, we found that both endogenous and overexpressed ERdj4 associate with the integral membrane protein, Derlin-1. Our findings now directly link ERdj4 to the ERAD machinery and suggest a model in which ERjd4 could help recruit clients from throughout the ER to ERAD sites.

Changes in BiP availability reveal hypersensitivity to acute endoplasmic reticulum stress in cells expressing mutant huntingtin.

Huntington's disease (HD) is caused by expanded glutamine repeats within the huntingtin (Htt) protein. Mutant Htt (mHtt) in the cytoplasm has been linked to induction of the luminal endoplasmic reticulum (ER) stress pathway, the unfolded protein response (UPR). How mHtt impacts the susceptibility of the ER lumen to stress remains poorly understood. To investigate molecular differences in the ER in cells expressing mHtt, we used live-cell imaging of a sensitive reporter of the misfolded secretory protein burden, GFP fused to the ER chaperone BiP (also known as GRP78), which decreases in mobility as it binds increasing amounts of misfolded proteins. Striatal neurons expressing full-length mHtt showed no differences in BiP-GFP mobility and no evidence of UPR activation compared with wild-type cells at steady state. However, mHtt-expressing cells were acutely sensitive to misfolded secretory proteins. Treatment with ER stressors, tunicamycin or DTT, rapidly decreased BiP-GFP mobility in mHtt striatal cells and accelerated UPR activation compared with wild-type cells. mHtt-expressing cells exhibited decreased misfolded protein flux as a result of ER associated degradation (ERAD) dysfunction. Furthermore, UPR-adapted mHtt cells succumbed to misfolded protein stresses that could be tolerated by adapted wild-type cells. Thus, mHtt expression impairs misfolded secretory protein turnover, decreases the ER stress threshold, and increases cell vulnerability to insults.

Cysteineless non-glycosylated monomeric blue fluorescent protein, secBFP2, for studies in the eukaryotic secretory pathway.

Fluorescent protein (FP) technologies suitable for use within the eukaryotic secretory pathway are essential for live cell and protein dynamic studies. Localization of FPs within the endoplasmic reticulum (ER) lumen has potentially significant consequences for FP function. All FPs are resident cytoplasmic proteins and have rarely been evolved for the chemically distinct environment of the ER lumen. In contrast to the cytoplasm, the ER lumen is oxidizing and the site where secretory proteins are post-translationally modified by disulfide bond formation and N-glycosylation on select asparagine residues. Cysteine residues and N-linked glycosylation consensus sequences were identified within many commonly utilized FPs. Here, we report mTagBFP is post-translationally modified when localized to the ER lumen. Our findings suggest these modifications can grossly affect the sensitivity and reliability of FP tools within the secretory pathway. To optimize tools for studying events in this important intracellular environment, we modified mTagBFP by mutating its cysteines and consensus N-glycosylation sites. We report successful creation of a secretory pathway-optimized blue FP, secBFP2.