In budding yeast, cell cycle progression and ribosome biogenesis are dependent on plasma membrane growth, which ensures that events of cell growth are coordinated with each other and with the cell cycle. However, the signals that link the cell cycle and ribosome biogenesis to membrane growth are poorly understood. Here we used proteome-wide mass spectrometry to systematically discover signals associated with membrane growth. The results suggest that membrane trafficking events required for membrane growth generate sphingolipid-dependent signals. A conserved signaling network appears to play an essential role in signaling by responding to delivery of sphingolipids to the plasma membrane. In addition, sphingolipid-dependent signals control phosphorylation of protein kinase C (Pkc1), which plays an essential role in the pathways that link the cell cycle and ribosome biogenesis to membrane growth. Together these discoveries provide new clues as to how growth-­dependent signals control cell growth and the cell cycle.

Several metabolic enzymes undergo reversible polymerization into macromolecular assemblies. The function of these assemblies is often unclear, but in some cases they regulate enzyme activity and metabolic homeostasis. The guanine nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) forms octamers that polymerize into helical chains. In mammalian cells, IMPDH filaments can associate into micron-length assemblies. Polymerization and enzyme activity are regulated in part by binding of purine nucleotides to an allosteric regulatory domain. ATP promotes octamer polymerization, whereas guanosine triphosphate (GTP) promotes a compact, inactive conformation whose ability to polymerize is unknown. Also unclear is whether polymerization directly alters IMPDH catalytic activity. To address this, we identified point mutants of human IMPDH2 that either prevent or promote polymerization. Unexpectedly, we found that polymerized and nonassembled forms of recombinant IMPDH have comparable catalytic activity, substrate affinity, and GTP sensitivity and validated this finding in cells. Electron microscopy revealed that substrates and allosteric nucleotides shift the equilibrium between active and inactive conformations in both the octamer and the filament. Unlike other metabolic filaments, which selectively stabilize active or inactive conformations, recombinant IMPDH filaments accommodate multiple states. These conformational states are finely tuned by substrate availability and purine balance, while polymerization may allow cooperative transitions between states.

The yeast bc1 complex (complex III) and cytochrome oxidase (complex IV) are mosaics of core subunits encoded by the mitochondrial genome and additional nuclear-encoded proteins imported from the cytosol. Both complexes build various supramolecular assemblies in the mitochondrial inner membrane. The formation of the individual complexes and their supercomplexes depends on the activity of dedicated assembly factors. We identified a so far uncharacterized mitochondrial protein (open reading frame YDR381C-A) as an important assembly factor for complex III, complex IV, and their supercomplexes. Therefore we named this protein Cox interacting (Coi) 1. Deletion of COI1 results in decreased respiratory growth, reduced membrane potential, and hampered respiration, as well as slow fermentative growth at low temperature. In addition, coi1Δ cells harbor reduced steady-state levels of subunits of complexes III and IV and of the assembled complexes and supercomplexes. Interaction of Coi1 with respiratory chain subunits seems transient, as it appears to be a stoichiometric subunit neither of complex III nor of complex IV. Collectively this work identifies a novel protein that plays a role in the assembly of the mitochondrial respiratory chain.

Synaptopathy underlying memory deficits in Alzheimer’s disease (AD) is increasingly thought to be instigated by toxic oligomers of the amyloid beta peptide (AβOs). Given the long latency and incomplete penetrance of AD dementia with respect to Aβ pathology, we hypothesized that factors present in the CNS may physiologically protect neurons from the deleterious impact of AβOs. Here we employed physically separated neuron–astrocyte cocultures to investigate potential non–cell autonomous neuroprotective factors influencing AβO toxicity. Neurons cultivated in the absence of an astrocyte feeder layer showed abundant AβO binding to dendritic processes and associated synapse deterioration. In contrast, neurons in the presence of astrocytes showed markedly reduced AβO binding and synaptopathy. Results identified the protective factors released by astrocytes as insulin and insulin-like growth factor-1 (IGF1). The protective mechanism involved release of newly bound AβOs into the extracellular medium dependent upon trafficking that was sensitive to exosome pathway inhibitors. Delaying insulin treatment led to AβO binding that was no longer releasable. The neuroprotective potential of astrocytes was itself sensitive to chronic AβO exposure, which reduced insulin/IGF1 expression. Our findings support the idea that physiological protection against synaptotoxic AβOs can be mediated by astrocyte-derived insulin/IGF1, but that this protection itself is vulnerable to AβO buildup.

Changes in cellular sterol species and concentrations can have profound effects on the transcriptional profile. In yeast, mutants defective in sterol biosynthesis show a wide range of changes in transcription, including a coinduction of anaerobic genes and ergosterol biosynthesis genes, biosynthesis of basic amino acids, and several stress genes. However the mechanisms underlying these changes are unknown. We identified mutations in the SAGA complex, a coactivator of transcription, which abrogate the ability to carry out most of these sterol-dependent transcriptional changes. In the erg3 mutant, the SAGA complex increases its occupancy time on many of the induced ergosterol and anaerobic gene promoters, increases its association with several relevant transcription factors and the SWI/SNF chromatin remodeling complex, and surprisingly, associates with an endocytic protein, Rvs167p, suggesting a moonlighting function for this protein in the sterol-regulated induction of the heat shock protein, HSP42 and HSP102, mRNAs.

The endothelium serves as a protective semipermeable barrier in blood vessels and lymphatic vessels. Leukocytes and pathogens can pass directly through the endothelium by opening holes in endothelial cells, known as transcellular tunnels, which are formed by contact and self-fusion of the apical and basal plasma membranes. Here we test the hypothesis that the actin cytoskeleton is the primary barrier to transcellular tunnel formation using a combination of atomic force microscopy and fluorescence microscopy of live cells. We find that localized mechanical forces are sufficient to induce the formation of transcellular tunnels in human umbilical vein endothelial cells (HUVECs). When HUVECs are exposed to the bacterial toxin called epidermal cell differentiation inhibitor (EDIN), which can induce spontaneous transcellular tunnels, less mechanical work is required to form tunnels due to the reduced cytoskeletal stiffness and thickness of these cells, similarly to the effects of a Rho-associated protein kinase (ROCK) inhibitor. We also observe actin enrichment in response to mechanical indentation that is reduced in cells exposed to the bacterial toxin. Our study shows that the actin cytoskeleton of endothelial cells provides both passive and active resistance against transcellular tunnel formation, serving as a mechanical barrier that can be overcome by mechanical force as well as disruption of the cytoskeleton.

Despite the importance of a cell’s ability to sense and respond to mechanical force, the molecular mechanisms by which physical cues are converted to cell-instructive chemical information to influence cell behaviors remain to be elucidated. Exposure of cultured fibroblasts to uniaxial cyclic stretch results in an actin stress fiber reinforcement response that stabilizes the actin cytoskeleton. p38 MAPK signaling is activated in response to stretch, and inhibition of p38 MAPK abrogates stretch-induced cytoskeletal reorganization. Here we show that the small heat shock protein HspB1 (hsp25/27) is phosphorylated in stretch-stimulated mouse fibroblasts via a p38 MAPK-dependent mechanism. Phosphorylated HspB1 is recruited to the actin cytoskeleton, displaying prominent accumulation on actin “comet tails” that emanate from focal adhesions in stretch-stimulated cells. Site-directed mutagenesis to block HspB1 phosphorylation inhibits the protein’s cytoskeletal recruitment in response to mechanical stimulation. HspB1-null cells, generated by CRISPR/Cas9 nuclease genome editing, display an abrogated stretch-stimulated actin reinforcement response and increased cell migration. HspB1 is recruited to sites of increased traction force in cells geometrically constrained on micropatterned substrates. Our findings elucidate a molecular pathway by which a mechanical signal is transduced via activation of p38 MAPK to influence actin remodeling and cell migration via a zyxin-independent process.

The dense core vesicles (DCVs) of neuroendocrine cells are a rich source of bioactive molecules such as peptides, hormones, and neurotransmitters, but relatively little is known about how they are formed. Using fractionation profiling, a method that combines subcellular fractionation with mass spectrometry, we identified ∼1200 proteins in PC12 cell vesicle-enriched fractions, with DCV-associated proteins showing distinct profiles from proteins associated with other types of vesicles. To investigate the role of clathrin in DCV biogenesis, we stably transduced PC12 cells with an inducible short hairpin RNA targeting clathrin heavy chain, resulting in ∼85% protein loss. DCVs could still be observed in the cells by electron microscopy, but mature profiles were approximately fourfold less abundant than in mock-treated cells. By quantitative mass spectrometry, DCV-associated proteins were found to be reduced approximately twofold in clathrin-depleted cells as a whole and approximately fivefold in vesicle-enriched fractions. Our combined data sets enabled us to identify new candidate DCV components. Secretion assays revealed that clathrin depletion causes a near-complete block in secretagogue-induced exocytosis. Taken together, our data indicate that clathrin has a function in DCV biogenesis beyond its established role in removing unwanted proteins from the immature vesicle.

Extended coiled-coil proteins of the golgin family play prominent roles in maintaining the structure and function of the Golgi complex. Here we further investigate the golgin protein Coy1 and document its function in retrograde transport between early Golgi compartments. Cells that lack Coy1 displayed a reduced half-life of the Och1 mannosyltransferase, an established cargo of intra-Golgi retrograde transport. Combining the coy1Δ mutation with deletions in other putative retrograde golgins (sgm1Δ and rud3Δ) caused strong glycosylation and growth defects and reduced membrane association of the conserved oligomeric Golgi (COG) complex. In contrast, overexpression of COY1 inhibited the growth of mutant strains deficient in fusion activity at the Golgi (sed5-1 and sly1-ts). To map Coy1 protein interactions, coimmunoprecipitation experiments revealed an association with the COG complex and with intra-Golgi SNARE proteins. These physical interactions are direct, as Coy1 was efficiently captured in vitro by Lobe A of the COG complex and the purified SNARE proteins Gos1, Sed5, and Sft1. Thus our genetic, in vivo, and biochemical data indicate a role for Coy1 in regulating COG complex-dependent fusion of retrograde-directed COPI vesicles.

Delivery of biomolecules to the correct subcellular locales is critical for proper physiological function. To that end, we have previously determined that type I and II transforming growth factor beta (TGF-β) receptors (TβRI and TβRII, respectively) localize to the basolateral domain in polarized epithelia. While TβRII targeting was shown to be regulated by sequences between amino acids 529 and 538, the analogous region(s) within TβRI is unknown. To address that question, sequential cytoplasmic TβRI truncations and point mutations identified a targeting motif between residues 158 and 163 (VxxEED) required for basolateral TβRI expression. Further studies documented that receptor internalization, down-regulation, direct recycling, or Smad signaling were unaffected by motif mutations that caused TβRI mislocalization. However, inclusion of amino acids 148–217 containing the targeting motif was able to direct basolateral expression of the apically sorted nerve growth factor receptor (NGFR, p75; extracellular and transmembrane regions) in a dominant manner. Finally, coexpression of apically targeted type I and type II TGF-β receptors mediated Smad3 signaling from the apical membrane of polarized epithelial cells. These findings demonstrate that the absence of apical TGF-β signaling in normal epithelia is primarily a reflection of domain-specific receptor expression and not an inability to couple with the signaling machinery.

One proposed mechanism of cellular aging is the gradual loss of certain cellular components that are insufficiently renewed. In an earlier study, multidrug resistance transporters (MDRs) were postulated to be such aging determinants during the yeast replicative life span (RLS). Aged MDR proteins were asymmetrically retained by the aging mother cell and did not diffuse freely into the bud, whereas newly synthesized MDR proteins were thought to be deposited mostly in the bud before cytokinesis. In this study, we further demonstrate the proposed age asymmetry of MDR proteins in dividing yeast cells and investigate the mechanism that controls diffusive properties of MDR proteins to maintain this asymmetry. We found that long-chain sphingolipids, but not the septin/endoplasmic reticulum–based membrane diffusion barrier, are important for restricting MDR diffusion. Depletion of sphingolipids or shortening of their long acyl chains resulted in an increase in the lateral mobility of MDR proteins, causing aged MDR protein in the mother cell to enter the bud. We used a mathematical model to understand the effect of diminished MDR age asymmetry on yeast cell aging, the result of which was qualitatively consistent with the observed RLS shortening in sphingolipid mutants.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane protein expressed on microglia within the brain. Several rare mutations in TREM2 cause an early-onset form of neurodegeneration when inherited homozygously. Here we investigate how these mutations affect the intracellular transport of TREM2. We find that most pathogenic TREM2 mutant proteins fail to undergo normal maturation in the Golgi complex and show markedly reduced cell-surface expression. Prior research has suggested that two such mutants are retained in the endoplasmic reticulum (ER), but we find, using a cell-free coat protein complex II (COPII) vesicle budding reaction, that mutant TREM2 is exported efficiently from the ER. In addition, mutant TREM2 becomes sensitive to cleavage by endoglycosidase D under conditions that inhibit recycling to the ER, indicating that it normally reaches a post-ER compartment. Maturation-defective TREM2 mutants are also efficiently bound by a lectin that recognizes O-glycans added in the ER–Golgi intermediate compartment (ERGIC) and cis-Golgi cisterna. Finally, mutant TREM2 accumulates in the ERGIC in cells depleted of COPI. These results indicate that efficient ER export is not sufficient to enable normal cell-surface expression of TREM2. Moreover, our findings suggest that the ERGIC may play an underappreciated role as a quality-control center for mutant and/or malformed membrane proteins.

Carbon fixation in cyanobacteria makes a major contribution to the global carbon cycle. The cyanobacterial carboxysome is a proteinaceous microcompartment that protects and concentrates the carbon-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in a paracrystalline lattice, making it possible for these organisms to fix CO₂ from the atmosphere. The protein responsible for the organization of this lattice in beta-type carboxysomes of the freshwater cyanobacterium Synechococcus elongatus, CcmM, occurs in two isoforms thought to localize differentially within the carboxysome matrix. Here we use wide-field time-lapse and three-dimensional structured illumination microscopy (3D-SIM) to study the recruitment and localization of these two isoforms. We demonstrate that this superresolution technique is capable of distinguishing the localizations of the outer protein shell of the carboxysome and its internal cargo. We develop an automated analysis pipeline to analyze and quantify 3D-SIM images and generate a population-level description of the carboxysome shell protein, RuBisCO, and CcmM isoform localization. We find that both CcmM isoforms have similar spatial and temporal localization, prompting a revised model of the internal arrangement of the β-carboxysome.