During terminal differentiation, the global protein complement is remodeled, as epitomized by erythrocytes, whose cytosol is ~98% globin. The erythroid proteome undergoes a rapid transition at the reticulocyte stage; however, the mechanisms driving programmed elimination of preexisting cytosolic proteins are unclear. We found that a mutation in the murine Ube2o gene, which encodes a ubiquitin-conjugating enzyme induced during erythropoiesis, results in anemia. Proteomic analysis suggested that UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome. In particular, ribosome elimination, a hallmark of reticulocyte differentiation, was defective in Ube2o−/− mutants. UBE2O recognized ribosomal proteins and other substrates directly, targeting them to proteasomes for degradation. Thus, in reticulocytes, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.

Signal peptide peptidase (SPP) and the four homologous SPP‐like (SPPL) proteases constitute a family of intramembrane aspartyl proteases with selectivity for type II‐oriented transmembrane segments. Here, we analyse the physiological function of the orphan protease SPPL2c, previously considered to represent a non‐expressed pseudogene. We demonstrate proteolytic activity of SPPL2c towards selected tail‐anchored proteins. Despite shared ER localisation, SPPL2c and SPP exhibit distinct, though partially overlapping substrate spectra and inhibitory profiles, and are organised in different high molecular weight complexes. Interestingly, SPPL2c is specifically expressed in murine and human testis where it is primarily localised in spermatids. In mice, SPPL2c deficiency leads to a partial loss of elongated spermatids and reduced motility of mature spermatozoa, but preserved fertility. However, matings of male and female SPPL2c−/− mice exhibit reduced litter sizes. Using proteomics we identify the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA2)‐regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease in intracellular Ca2+ levels in elongated spermatids that likely contribute to the compromised male germ cell differentiation and function of SPPL2c−/− mice.

Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae open reading frames (ORFs). In 5,661 strains, we inserted an acceptor module after each ORF that can be efficiently replaced with tags or regulatory elements. We validated the library with targeted sequencing and tagged the proteome with bright fluorescent proteins to quantify the effect of heterologous transcription terminators on protein expression and to localize previously undetected proteins.

Tail-anchored (TA) proteins insert post-translationally into the endoplasmic reticulum (ER), the outer mitochondrial membrane (OMM) and peroxisomes. Whereas the GET pathway controls ER-targeting, no dedicated factors are known for OMM insertion, posing the question of how accuracy is achieved. The mitochondrial AAA-ATPase Msp1 removes mislocalized TA proteins from the OMM, but it is unclear, how Msp1 clients are targeted for degradation. Here we screened for factors involved in degradation of TA proteins mislocalized to mitochondria. We show that the ER-associated degradation (ERAD) E3 ubiquitin ligase Doa10 controls cytoplasmic level of Msp1 clients. Furthermore, we identified the uncharacterized OMM protein Fmp32 and the ectopically expressed subunit of the ER-mitochondria encounter structure (ERMES) complex Gem1 as native clients for Msp1 and Doa10. We propose that productive localization of TA proteins to the OMM is ensured by complex assembly, while orphan subunits are extracted by Msp1 and eventually degraded by Doa10.

Over the last two decades, a group of unusual proteases, so-called intramembrane proteases, have become increasingly recognized for their unique ability to cleave peptide bonds within cellular membranes. They are found in all kingdoms of life and fulfil versatile functions ranging from protein maturation, to activation of signalling molecules, to protein degradation. In this Cell Science at a Glance article and the accompanying poster, we focus on intramembrane proteases in mammalian cells. By comparing intramembrane proteases in different cellular organelles, we set out to review their functions within the context of the roles of individual cellular compartments. Additionally, we exemplify their mode of action in relation to known substrates by distinguishing cleavage events that promote degradation of substrate from those that release active domains from the membrane bilayer.

Pseudomonas aeruginosa has emerged as an important opportunistic human pathogen that is often highly resistant to eradication strategies, mediated in part by the formation of multicellular aggregates. Cellular aggregates may occur attached to a surface (biofilm), at the air-liquid interface (pellicle), or as suspended aggregates. Compared to surface attached communities, knowledge about the regulatory processes involved in the formation of suspended cell aggregates is still limited. We have recently described the SiaA/D signal transduction module that regulates macroscopic cell aggregation during growth with, or in the presence of the surfactant SDS. Targets for SiaA/D mediated regulation include the Psl polysaccharide, the CdrAB two-partner secretion system and the CupA fimbriae. While the global regulators c-di-GMP and RsmA are known to inversely coordinate cell aggregation and regulate the expression of several adhesins, their potential impact on the expression of the cupA operon remains unknown. Here, we investigated the function of SiaA (a putative ser/thr phosphatase) and SiaD (a di-guanylate cyclase) in cupA1 expression using transcriptional reporter fusions and qRT-PCR. These studies revealed a novel interaction between the RsmA posttranscriptional regulatory system and SiaA/D mediated macroscopic aggregation. The RsmA/rsmY/Z system was found to affect macroscopic aggregate formation in the presence of surfactant by impacting the stability of the cupA1 mRNA transcript and we reveal that RsmA directly binds to the cupA1 leader sequence in vitro. We further identified that transcription of the RsmA antagonist rsmZ is controlled in a SiaA/D dependent manner during growth with SDS. Finally, we found that the siaD transcript is also under regulatory control of RsmA and that overproduction of RsmA or the deletion of siaD results in decreased cellular cyclic di-guanosine monophosphate (c-di-GMP) levels quantified by a transcriptional reporter, demonstrating that SiaA/D connects c-di-GMP and RsmA/rsmY/Z signaling to reciprocally regulate cell aggregation in response to environmental conditions.

A growing body of evidence reveals the importance of high-quality teacher-child interactions in early childhood education and care (ECEC) for learning and the behavioral engagement of young children before they enter school (Mashburn et al., 2008;Ponitz, Rimm-Kaufman, Grimm, & Curby, 2009). In general, teachers' performance and effectiveness is seen a function of their behavior in classrooms and their interactions with students. In line with that, the "Teaching Through Interactions" framework (Hamre et al., 2013) assumes that the quality of interactions between children and adults is the primary mechanism to promote developmental gains. This paper therefore focuses on efforts to improve the quality of teacher-child-interactions in ECEC classrooms. According to the framework, interactions between teacher and child are distinguished in three domains, namely emotional support (ES), classroom organization (CO), and instructional support (IS). The observational measure Classroom Assessment Scoring System (CLASS Pre-K) was developed to assess the quality of teacher-child interactions in ECEC for these three domains based on this framework (Hamre et al., 2013;Pianta, La Paro, & Hamre, 2008a). Emotional support (ES) involves teacher sensitivity and their responsiveness to children's signals, as well as regard for student perspectives, interests, and motivation (Pianta et al., 2008a). Classroom organization (CO) comprises a variety of teacher-child interactions that help students to organize their behavior and attention related to classroom routines and academic goals (Hamre et al., 2013). A teacher who sets clear rules, organizes stable classroom routines, uses proactive approaches to discipline, and provides interesting and diversified activities contributes significantly to children's motivation and engagement in learning tasks (Pianta et al., 2008a). Instructionally supportive teachers (IS) offer a wide range of learning activities to enhance knowledge of concepts and language, relate new information to students' background knowledge and real-world experiences, and provide learning feedback that is immediate, corrective, and specific (Hamre et al., 2013;Pianta et al., 2008a). High levels of ES, CO or IS indicate that a teacher provides high-quality teacher-child interactions consistently throughout the day and during different activities (Hamre et al., 2013;Pianta et al., 2008a). Teacher-child interactions and child developmentEmpirical research has found consistent associations between the quality of teacher-child interactions measured with the CLASS and children's outcomes. The magnitude of this association is small (see Keys et al., 2013;Perlman et al., 2016). Recent research indicates that a certain quality threshold must be reached to affect students' school readiness skills (Hatfield, Burchinal, Pianta, & Sideris, 2016;Wieland, Ulvestad, Sachs, & Yoshikawa, 2013). Further, different quality domains of teacher-child interactions seem to be predictive for specific competencies of children in preschool and kinderga...

The pyrimidine core has been utilized extensively to construct kinase inhibitors, including eight FDA-approved drugs. Because the pyrimidine hinge-binding motif is accommodated by many human kinases, kinome-wide selectivity of resultant molecules can be poor. This liability was seen as an advantage since it is well tolerated by many understudied kinases. We hypothesized that nonexemplified aminopyrimidines bearing side chains from well-annotated pyrimidine-based inhibitors with off-target activity on understudied kinases would provide us with useful inhibitors of these lesser studied kinases. Our strategy paired mixing and matching the side chains from the 2- and 4-positions of the parent compounds with modifications at the 5-position of the pyrimidine core, which is situated near the gatekeeper residue of the binding pocket. Utilizing this approach, we imparted improved kinome-wide selectivity to most members of the resultant library. Importantly, we also identified potent biochemical and cell-active lead compounds for understudied kinases like DRAK1, BMP2K, and MARK3/4.