Empowering STEM students: A university-wide mentorship program fostering retention and belonging.

In the face of a challenging climate STEM (Science, Technology, Engineering and Mathematics) higher education that is resistant to Diversity, Equity, and Inclusion efforts aimed to increase and retain students from historically excluded groups (HEGs), there is a critical need for a support structure to ensure students from HEGs continue to be recruited retained. The Biology Undergraduate and Master's Mentorship Program (BUMMP) embodies this commitment to fostering scientific identity, efficacy, and a sense of belonging for first-generation and historically underserved undergraduate and master's students at UC San Diego. The mission of BUMMP is to cultivate a sense of belonging, instill confidence, and nurture a strong scientific identity amongst all its participants. At its core, the three pillars of BUMMP are (1) mentorship, (2) professional development, and (3) research. Quality mentorship is provided where students receive personal guidance from faculty, graduate students, postdocs, and industry leaders in navigating their career pathways. Complementing mentorship, BUMMP provides paid research opportunities and prioritizes professional development by offering workshops designed to enhance students' professional skills. These three pillars form the backbone of BUMMP, empowering students from all backgrounds and ensuring their retention and persistence in STEM. So far, we've served over 1350 mentees, collaborated with 809 mentors, and had over 180 mentees actively engaged in BUMMP-sponsored research activities. The primary focus of this paper is to provide a programmatic guideline for the three pillars of BUMMP: mentorship, professional development, and research. This will offer a blueprint for other institutions to establish similar mentorship programs. Additionally, the paper highlights the impact of the BUMMP program and surveyed mentees who have participated in the mentorship and research component of BUMMP. We showed that mentorship and research experience enhance students' sense of belonging, science identity, and science efficacy, which are key predictors of retention and persistence in pursuing a STEM career. Overall, BUMMP's expansive efforts have made a tremendous impact at UC San Diego and will continue to foster a community of future leaders who will be prepared to make meaningful contributions to the scientific community and beyond.

Proteotoxic stress and the ubiquitin proteasome system.

The ubiquitin proteasome system maintains protein homeostasis by regulating the breakdown of misfolded proteins, thereby preventing misfolded protein aggregates. The efficient elimination is vital for preventing damage to the cell by misfolded proteins, known as proteotoxic stress. Proteotoxic stress can lead to the collapse of protein homeostasis and can alter the function of the ubiquitin proteasome system. Conversely, impairment of the ubiquitin proteasome system can also cause proteotoxic stress and disrupt protein homeostasis. This review examines two impacts of proteotoxic stress, 1) disruptions to ubiquitin homeostasis (ubiquitin stress) and 2) disruptions to proteasome homeostasis (proteasome stress). Here, we provide a mechanistic description of the relationship between proteotoxic stress and the ubiquitin proteasome system. This relationship is illustrated by findings from several protein misfolding diseases, mainly neurodegenerative diseases, as well as from basic biology discoveries from yeast to mammals. In addition, we explore the importance of the ubiquitin proteasome system in endoplasmic reticulum quality control, and how proteotoxic stress at this organelle is alleviated. Finally, we highlight how cells utilize the ubiquitin proteasome system to adapt to proteotoxic stress and how the ubiquitin proteasome system can be genetically and pharmacologically manipulated to maintain protein homeostasis.

Juneteenth in STEMM and the barriers to equitable science.

We are 52 Black scientists. Here, we establish the context of Juneteenth in STEMM and discuss the barriers Black scientists face, the struggles they endure, and the lack of recognition they receive. We review racism's history in science and provide institutional-level solutions to reduce the burdens on Black scientists.

Yeast derlin Dfm1 employs a chaperone-like function to resolve misfolded membrane protein stress.

Protein aggregates are a common feature of diseased and aged cells. Membrane proteins comprise a quarter of the proteome, and yet, it is not well understood how aggregation of membrane proteins is regulated and what effects these aggregates can have on cellular health. We have determined in yeast that the derlin Dfm1 has a chaperone-like activity that influences misfolded membrane protein aggregation. We establish that this function of Dfm1 does not require recruitment of the ATPase Cdc48 and it is distinct from Dfm1's previously identified function in dislocating misfolded membrane proteins from the endoplasmic reticulum (ER) to the cytosol for degradation. Additionally, we assess the cellular impacts of misfolded membrane proteins in the absence of Dfm1 and determine that misfolded membrane proteins are toxic to cells in the absence of Dfm1 and cause disruptions to proteasomal and ubiquitin homeostasis.

The Role of the Rhomboid Superfamily in ER Protein Quality Control: From Mechanisms and Functions to Diseases.

The endoplasmic reticulum (ER) is an essential organelle in eukaryotic cells and is a major site for protein folding, modification, and lipid synthesis. Perturbations within the ER, such as protein misfolding and high demand for protein folding, lead to dysregulation of the ER protein quality control network and ER stress. Recently, the rhomboid superfamily has emerged as a critical player in ER protein quality control because it has diverse cellular functions, including ER-associated degradation (ERAD), endosome Golgi-associated degradation (EGAD), and ER preemptive quality control (ERpQC). This breadth of function both illustrates the importance of the rhomboid superfamily in health and diseases and emphasizes the necessity of understanding their mechanisms of action. Because dysregulation of rhomboid proteins has been implicated in various diseases, such as neurological disorders and cancers, they represent promising potential therapeutic drug targets. This review provides a comprehensive account of the various roles of rhomboid proteins in the context of ER protein quality control and discusses their significance in health and disease.

An ERAD-independent role for rhomboid pseudoprotease Dfm1 in mediating sphingolipid homeostasis.

Nearly one-third of nascent proteins are initially targeted to the endoplasmic reticulum (ER), where they are correctly folded and assembled before being delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated degradation (ERAD) removes these client proteins from the ER membrane to the cytosol in a process known as retrotranslocation. Our previous work demonstrated that rhomboid pseudoprotease Dfm1 is involved in the retrotranslocation of ubiquitinated membrane integral ERAD substrates. Herein, we found that Dfm1 associates with the SPOTS complex, which is composed of serine palmitoyltransferase (SPT) enzymes and accessory components that are critical for catalyzing the first rate-limiting step of the sphingolipid biosynthesis pathway. Furthermore, Dfm1 employs an ERAD-independent role for facilitating the ER export and endosome- and Golgi-associated degradation (EGAD) of Orm2, which is a major antagonist of SPT activity. Given that the accumulation of human Orm2 homologs, ORMDLs, is associated with various pathologies, our study serves as a molecular foothold for understanding how dysregulation of sphingolipid metabolism leads to various diseases.

Quality Control: Maintaining molecular order and preventing cellular chaos.

We asked experts from different fields-from genome maintenance and proteostasis to organelle degradation via ubiquitin and autophagy-"What does quality control mean to you?" Despite their diverse backgrounds, they converge on and discuss the importance of continuous quality control at all levels, context, communication, timing, decisions on whether to repair or remove, and the significance of dysregulated quality control in disease.

Decoding Transcription Regulatory Mechanisms Associated with Coccidioides immitis Phase Transition Using Total RNA.

New or emerging infectious diseases are commonly caused by pathogens that cannot be readily manipulated or studied under common laboratory conditions. These limitations hinder standard experimental approaches and our abilities to define the fundamental molecular mechanisms underlying pathogenesis. The advance of capped small RNA sequencing (csRNA-seq) now enables genome-wide mapping of actively initiated transcripts from genes and other regulatory transcribed start regions (TSRs) such as enhancers at a precise moment from total RNA. As RNA is nonpathogenic and can be readily isolated from inactivated infectious samples, csRNA-seq can detect acute changes in gene regulation within or in response to a pathogen with remarkable sensitivity under common laboratory conditions. Studying valley fever (coccidioidomycosis), an emerging endemic fungal infection that increasingly impacts livestock, pet, and human health, we show how csRNA-seq can unravel transcriptional programs driving pathogenesis. Performing csRNA-seq on RNA isolated from different stages of the valley fever pathogen Coccidioides immitis revealed alternative promoter usage, connected cis-regulatory domains, and a WOPR family transcription factor, which are known regulators of virulence in other fungi, as being critical for pathogenic growth. We further demonstrate that a C. immitis WOPR homologue, CIMG_02671, activates transcription in a WOPR motif-dependent manner. Collectively, these findings provide novel insights into valley fever pathogenesis and provide a proof of principle for csRNA-seq as a powerful means to determine the genes, regulatory mechanisms, and transcription factors that control the pathogenesis of highly infectious agents. IMPORTANCE Infectious pathogens like airborne viruses or fungal spores are difficult to study; they require high-containment facilities, special equipment, and expertise. As such, establishing approaches such as genome editing or other means to identify the factors and mechanisms underlying caused diseases, and, thus, promising drug targets, is costly and time-intensive. These obstacles particularly hinder the analysis of new, emerging, or rare infectious diseases. We recently developed a method termed capped small RNA sequencing (csRNA-seq) that enables capturing acute changes in active gene expression from total RNA. Prior to csRNA-seq, such an analysis was possible only by using living cells or nuclei, in which pathogens are highly infectious. The process of RNA purification, however, inactivates pathogens and thus enables the analysis of gene expression during disease progression under standard laboratory conditions. As a proof of principle, here, we use csRNA-seq to unravel the gene regulatory programs and factors likely critical for the pathogenesis of valley fever, an emerging endemic fungal infection that increasingly impacts livestock, pet, and human health.

Derlin rhomboid pseudoproteases employ substrate engagement and lipid distortion to enable the retrotranslocation of ERAD membrane substrates.

Nearly one-third of proteins are initially targeted to the endoplasmic reticulum (ER) membrane, where they are correctly folded and then delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated degradation (ERAD) moves these clients from the ER membrane to the cytosol, a process known as retrotranslocation. Our recent work in Saccharomyces cerevisiae reveals a derlin rhomboid pseudoprotease, Dfm1, is involved in the retrotranslocation of ubiquitinated ERAD membrane substrates. In this study, we identify conserved residues of Dfm1 that are critical for retrotranslocation. We find several retrotranslocation-deficient Loop 1 mutants that display impaired binding to membrane substrates. Furthermore, Dfm1 possesses lipid thinning function to facilitate in the removal of ER membrane substrates, and this feature is conserved in its human homolog, Derlin-1, further implicating that derlin-mediated retrotranslocation is a well-conserved process.

Assays for studying normal versus suppressive ERAD-associated retrotranslocation pathways in yeast.

In S. cerevisiae, we identified rhomboid pseudoprotease Dfm1 as the major mediator for removing or retrotranslocating misfolded membrane substrates from the ER (endoplasmic reticulum). Long-standing challenges with rapid suppression of dfm1-null cells have limited the biochemical study of Dfm1's role in ER protein quality control. Here, we provide a protocol for the generation and handling of dfm1-null cells and procedures for studying normal vs. suppressive alternative retrotranslocation pathways. Our methods can be utilized to study other components involved in retrotranslocation. For complete information on the generation and use of this protocol, please refer to Neal et al. (2017, 2018); Neal et al. (2019); Neal et al. (2020).

Impaired phosphatidylethanolamine metabolism activates a reversible stress response that detects and resolves mutant mitochondrial precursors.

Phosphatidylethanolamine (PE) made in mitochondria has long been recognized as an important precursor for phosphatidylcholine production that occurs in the endoplasmic reticulum (ER). Recently, the strict mitochondrial localization of the enzyme that makes PE in the mitochondrion, phosphatidylserine decarboxylase 1 (Psd1), was questioned. Since a dual localization of Psd1 to the ER would have far-reaching implications, we initiated our study to independently re-assess the subcellular distribution of Psd1. Our results support the unavoidable conclusion that the vast majority, if not all, of functional Psd1 resides in the mitochondrion. Through our efforts, we discovered that mutant forms of Psd1 that impair a self-processing step needed for it to become functional are dually localized to the ER when expressed in a PE-limiting environment. We conclude that severely impaired cellular PE metabolism provokes an ER-assisted adaptive response that is capable of identifying and resolving nonfunctional mitochondrial precursors.

Inner-nuclear-membrane-associated degradation employs Dfm1-independent retrotranslocation and alleviates misfolded transmembrane-protein toxicity.

Before their delivery to and degradation by the 26S proteasome, misfolded transmembrane proteins of the endoplasmic reticulum (ER) and inner-nuclear membrane (INM) must be extracted from lipid bilayers. This extraction process, known as retrotranslocation, requires both quality-control E3 ubiquitin ligases and dislocation factors that diminish the energetic cost of dislodging the transmembrane segments of a protein. Recently, we showed that retrotranslocation of all ER transmembrane proteins requires the Dfm1 rhomboid pseudoprotease. However, we did not investigate whether Dfm1 also mediated retrotranslocation of transmembrane substrates in the INM, which is contiguous with the ER but functionally separated from it by nucleoporins. Here, we show that canonical retrotranslocation occurs during INM-associated degradation (INMAD) but proceeds independently of Dfm1. Despite this independence, ER-associated degradation (ERAD)-M and INMAD cooperate to mitigate proteotoxicity. We show a novel misfolded-transmembrane-protein toxicity that elicits genetic suppression, demonstrating the cell's ability to tolerate a toxic burden of misfolded transmembrane proteins without functional INMAD or ERAD-M. This strikingly contrasted the suppression of the dfm1Δ null, which leads to the resumption of ERAD-M through HRD-complex remodeling. Thus, we conclude that INM retrotranslocation proceeds through a novel, private channel that can be studied by virtue of its role in alleviating membrane-associated proteotoxicity.

HRD Complex Self-Remodeling Enables a Novel Route of Membrane Protein Retrotranslocation.

ER-associated degradation (ERAD) targets misfolded ER proteins for degradation. Retrotranslocation, a key feature of ERAD, entails removal of ubiquitinated substrates into the cytosol for proteasomal destruction. Recently, it has been shown that the Hrd1 E3 ligase forms a retrotranslocation channel for luminal (ERAD-L) substrates. Conversely, our studies found that integral membrane (ERAD-M) substrates exit the ER through a distinct pathway mediated by the Dfm1 rhomboid protein. Those studies also revealed a second, Hrd1-dependent pathway of ERAD-M retrotranslocation can arise in dfm1Δ null. Here we show that, in the dfm1Δ null, the HRD complex undergoes remodeling to a form that mediates ERAD-M retrotranslocation. Specifically, Hrd1's normally present stochiometric partner Hrd3 is efficiently removed during suppressive remodeling, allowing Hrd1 to function in this novel capacity. Neither Hrd1 autoubiquitination nor its cytosolic domain is required for suppressive ERAD-M retrotranslocation. Thus, the HRD complex displays remarkable functional flexibility in response to ER stress.

The role of rhomboid superfamily members in protein homeostasis: Mechanistic insight and physiological implications.

Cells are equipped with protein quality control pathways in order to maintain a healthy proteome; a process known as protein homeostasis. Dysfunction in protein homeostasis leads to the development of many diseases that are associated with proteinopathies. Recently, the rhomboid superfamily has attracted much attention concerning their involvement in protein homeostasis. While their functional role has become much clearer in the last few years, their systemic significance in mammals remains elusive. Here we delineate the current knowledge of rhomboids in protein quality control and how these functions are integrated at the organismal level.

Assays for protein retrotranslocation in ERAD.

Elimination of misfolded proteins by endoplasmic reticulum (ER)-associated protein degradation (ERAD) ensures that proteins proceeding through the secretory pathway are correctly folded and processed, which is critical to minimize ER stress. All ERAD pathways include a protein translocation process termed retrotranslocation, in which ubiquitinated misfolded substrates are extracted from the ER and degraded by the cytosolic 26S proteasome. Despite being integral to ERAD, the retrotranslocation process has been largely obscure. Recently, an explosion of discoveries has provided key mechanistic insights into this novel route of protein transport. These advances were facilitated by the development of in vitro and in vivo assays that utilize components from the yeast Saccharomyces cerevisiae. The assays permit detailed study of the distinct steps in ERAD-linked retrotranslocation, including ubiquitination of selected ERAD substrates, substrate removal from the ER, maintenance of cytosolic substrate solubility in the cytosol, and substrate degradation. Here we provide detailed protocols for these assays that pertain to work on retrotranslocation of integral membrane proteins (ERAD-M substrates), with the expectation that these approaches can be adapted for many related biochemical processes.

The Dfm1 Derlin Is Required for ERAD Retrotranslocation of Integral Membrane Proteins.

Endoplasmic reticulum (ER)-associated degradation (ERAD) removes misfolded proteins from the ER membrane and lumen by the ubiquitin-proteasome pathway. Retrotranslocation of ubiquitinated substrates to the cytosol is a universal feature of ERAD that requires the Cdc48 AAA-ATPase. Despite intense efforts, the mechanism of ER exit, particularly for integral membrane (ERAD-M) substrates, has remained unclear. Using a self-ubiquitinating substrate (SUS), which undergoes normal retrotranslocation independently of known ERAD factors, and the new SPOCK (single plate orf compendium kit) micro-library to query all yeast genes, we found the rhomboid derlin Dfm1 was required for retrotranslocation of both HRD and DOA ERAD pathway integral membrane substrates. Dfm1 recruited Cdc48 to the ER membrane with its unique SHP motifs, and it catalyzed substrate extraction through its conserved rhomboid motifs. Surprisingly, dfm1Δ can undergo rapid suppression, restoring wild-type ERAD-M. This unexpected suppression explained earlier studies ruling out Dfm1, and it revealed an ancillary ERAD-M retrotranslocation pathway requiring Hrd1.

Osm1 facilitates the transfer of electrons from Erv1 to fumarate in the redox-regulated import pathway in the mitochondrial intermembrane space.

Prokaryotes have aerobic and anaerobic electron acceptors for oxidative folding of periplasmic proteins. The mitochondrial intermembrane space has an analogous pathway with the oxidoreductase Mia40 and sulfhydryl oxidase Erv1, termed the mitochondrial intermembrane space assembly (MIA) pathway. The aerobic electron acceptors include oxygen and cytochrome c, but an acceptor that can function under anaerobic conditions has not been identified. Here we show that the fumarate reductase Osm1, which facilitates electron transfer from fumarate to succinate, fills this gap as a new electron acceptor. In addition to microsomes, Osm1 localizes to the mitochondrial intermembrane space and assembles with Erv1 in a complex. In reconstitution studies with reduced Tim13, Mia40, and Erv1, the addition of Osm1 and fumarate completes the disulfide exchange pathway that results in Tim13 oxidation. From in vitro import assays, mitochondria lacking Osm1 display decreased import of MIA substrates, Cmc1 and Tim10. Comparative reconstitution assays support that the Osm1/fumarate couple accepts electrons with similar efficiency to cytochrome c and that the cell has strategies to coordinate expression of the terminal electron acceptors. Thus Osm1/fumarate is a new electron acceptor couple in the mitochondrial intermembrane space that seems to function in both aerobic and anaerobic conditions.

A Cdc48 "Retrochaperone" Function Is Required for the Solubility of Retrotranslocated, Integral Membrane Endoplasmic Reticulum-associated Degradation (ERAD-M) Substrates.

A surprising feature of endoplasmic reticulum (ER)-associated degradation (ERAD) is the movement, or retrotranslocation, of ubiquitinated substrates from the ER lumen or membrane to the cytosol where they are degraded by the 26S proteasome. Multispanning ER membrane proteins, called ERAD-M substrates, are retrotranslocated to the cytosol as full-length intermediates during ERAD, and we have investigated how they maintain substrate solubility. Using an in vivo assay, we show that retrotranslocated ERAD-M substrates are moved to the cytoplasm as part of the normal ERAD pathway, where they are part of a solely proteinaceous complex. Using proteomics and direct biochemical confirmation, we found that Cdc48 serves as a critical "retrochaperone" for these ERAD-M substrates. Cdc48 binding to retrotranslocated, ubiquitinated ERAD-M substrates is required for their solubility; removal of the polyubiquitin chains or competition for binding by addition of free polyubiquitin liberated Cdc48 from retrotranslocated proteins and rendered them insoluble. All components of the canonical Cdc48 complex Cdc48-Npl4-Ufd1 were present in solubilized ERAD-M substrates. This function of the complex was observed for both HRD and DOA pathway substrates. Thus, in addition to the long known ATP-dependent extraction of ERAD substrates during retrotranslocation, the Cdc48 complex is generally and critically needed for the solubility of retrotranslocated ERAD-M intermediates.