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Secretory proteins are critically dependent on the correct processing of their signal sequence by the signal peptidase complex (SPC). This step, which is essential for the proper folding and localization of proteins in eukaryotic cells, is still not fully understood. In eukaryotes, the SPC comprises four evolutionarily conserved membrane subunits (Spc1-3 and Sec11). Here, we investigated the role of Spc2, examining SPC cleavage efficiency on various models and natural signal sequences in yeast cells depleted of or with mutations in Spc2. Our data show that discrimination between substrates and identification of the cleavage site by SPC is compromised when Spc2 is absent or mutated. Molecular dynamics simulation of the yeast SPC AlphaFold2-Multimer model indicates that membrane thinning at the center of SPC is reduced without Spc2, suggesting a molecular explanation for the altered substrate recognition properties of SPC lacking Spc2. These results provide new insights into the molecular mechanisms by which SPC governs protein biogenesis., (© 2024 Chung et al.)

Mogk and den Brave discuss exciting results from a comprehensive screen of heat shock response components in yeast, published in this issue by Pincus and colleagues (https://doi.org/10.1083/jcb.202401082). Their work reveals modulatory regulatory loops that fine-tune the timing of the shutdown of this highly conserved pathway., (© 2024 Mogk and den Brave.)

Efficient import of nuclear-encoded proteins into mitochondria is crucial for proper mitochondrial function. The conserved translation factor eIF5A binds ribosomes, alleviating stalling at polyproline-encoding sequences. eIF5A impacts mitochondrial function across species, though the precise molecular mechanism is unclear. We found that eIF5A depletion in yeast reduces the translation and levels of the TCA cycle and oxidative phosphorylation proteins. Loss of eIF5A causes mitoprotein precursors to accumulate in the cytosol and triggers a mitochondrial import stress response. We identify an essential polyproline protein as a direct target of eIF5A: the mitochondrial inner membrane protein and translocase component Tim50. Thus, eIF5A controls mitochondrial protein import by alleviating ribosome stalling along Tim50 mRNA at the mitochondrial surface. Removal of polyprolines from Tim50 partially rescues the mitochondrial import stress response and translation of oxidative phosphorylation genes. Overall, our findings elucidate how eIF5A impacts the mitochondrial function by promoting efficient translation and reducing ribosome stalling of co-translationally imported proteins, thereby positively impacting the mitochondrial import process., (© 2024 Barba-Aliaga et al.)

Cells maintain homeostasis via dynamic regulation of stress response pathways. Stress pathways transiently induce response regulons via negative feedback loops, but the extent to which individual genes provide feedback has not been comprehensively measured for any pathway. Here, we disrupted the induction of each gene in the Saccharomyces cerevisiae heat shock response (HSR) and quantified cell growth and HSR dynamics following heat shock. The screen revealed a core feedback loop governing the expression of the chaperone Hsp70 reinforced by an auxiliary feedback loop controlling Hsp70 subcellular localization. Mathematical modeling and live imaging demonstrated that multiple HSR targets converge to promote Hsp70 nuclear localization via its release from cytosolic condensates. Following ethanol stress, a distinct set of factors similarly converged on Hsp70, suggesting that nonredundant subsets of the HSR regulon confer feedback under different conditions. Flexible spatiotemporal feedback loops may broadly organize stress response regulons and expand their adaptive capacity., (© 2024 Garde et al.)

As a consequence of hypoosmotic shock, yeast cells swell rapidly and increase the surface area by ∼20% in 20 s. Approximately, 35% of this surface increase is mediated by the ER-plasma membrane contact sites, specifically the tricalbins, which are required for the delivery of both lipids and the GPI-anchored protein Crh2 from the cortical ER to the plasma membrane. Therefore, we propose a new function for the tricalbins: mediating the fusion of the ER to the plasma membrane at contact sites. This proposed fusion is triggered by calcium influx via the stretch-gated channel Cch1 and is supported by the anoctamin Ist2., (© 2024 Smith et al.)

The outer mitochondrial membrane (OMM) creates a boundary that imports most of the mitochondrial proteome while removing extraneous or damaged proteins. How the OMM senses aberrant proteins and remodels to maintain OMM integrity remains unresolved. Previously, we identified a mitochondrial remodeling mechanism called the mitochondrial-derived compartment (MDC) that removes a subset of the mitochondrial proteome. Here, we show that MDCs specifically sequester proteins localized only at the OMM, providing an explanation for how select mitochondrial proteins are incorporated into MDCs. Remarkably, selective sorting into MDCs also occurs within the OMM, as subunits of the translocase of the outer membrane (TOM) complex are excluded from MDCs unless assembly of the TOM complex is impaired. Considering that overloading the OMM with mitochondrial membrane proteins or mistargeted tail-anchored membrane proteins induces MDCs to form and sequester these proteins, we propose that one functional role of MDCs is to create an OMM-enriched trap that segregates and sequesters excess proteins from the mitochondrial surface., (© 2024 Wilson et al.)

Core histones, synthesized and processed in the cytoplasm, must be chaperoned as they are transported into the nucleus for nucleosome assembly. The importin Kap114 transports H2A-H2B into the yeast nucleus, where RanGTP facilitates histone release. Kap114 and H2A-H2B also bind the histone chaperone Nap1, but how Nap1 and Kap114 cooperate in transport and nucleosome assembly remains unclear. Here, biochemical and structural analyses show that Kap114, H2A-H2B, and a Nap1 dimer (Nap12) associate in the absence and presence of RanGTP to form equimolar complexes. A previous study had shown that RanGTP reduces Kap114's ability to chaperone H2A-H2B, but a new cryo-EM structure of the Nap12•H2A-H2B•Kap114•RanGTP complex explains how both Kap114 and Nap12 interact with H2A-H2B, restoring its chaperoning within the assembly while effectively depositing it into nucleosomes. Together, our results suggest that Kap114 and Nap12 provide a sheltered path that facilitates the transfer of H2A-H2B from Kap114 to Nap12, ultimately directing its specific deposition into nucleosomes., (© 2024 Fung et al.)

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is a low-abundance signaling lipid that plays crucial roles in various cellular processes, including endolysosomal system structure/function, stress response, and cell cycle regulation. PI(3,5)P2 synthesis increases in response to environmental stimuli, yet its behavior in cycling cells under basal conditions remains elusive. Here, we analyzed spatiotemporal changes in PI(3,5)P2 levels during the cell cycle of S. cerevisiae. We found that PI(3,5)P2 accumulates on the vacuole in the daughter cell while it disappears from the vacuole in the mother cell during mitosis. Concomitant with the changes in PI(3,5)P2 distribution, the daughter vacuole became more acidic, whereas the acidity of the mother vacuole decreased during mitosis. Our data further showed that both PI(3,5)P2 and the PI(3,5)P2 effector protein Atg18 are determinants of vacuolar-pH asymmetry and acidity. Our work, thus, identifies PI(3,5)P2 as a key factor for the establishment of vacuolar-pH asymmetry, providing insights into how the mother cell ages while the daughter cell is rejuvenated., (© 2024 Huda et al.)

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a phospholipid essential for plasma membrane functions, but its two-dimensional distribution is not clear. Here, we compared the result of sodium dodecyl sulfate-treated freeze-fracture replica labeling (SDS-FRL) of quick-frozen cells with the actual PtdIns(4,5)P2 content and the results obtained by fluorescence biosensor and by labeling of chemically-fixed membranes. In yeast, enrichment of PtdIns(4,5)P2 in the membrane compartment of Can1 (MCC)/eisosome, especially in the curved MCC/eisosome, was evident by SDS-FRL, but not by fluorescence biosensor, GFP-PLC1δ-PH. PtdIns(4,5)P2 remaining after acute ATP depletion and in the stationary phase, 30.0% and 56.6% of the control level, respectively, was not detectable by fluorescence biosensor, whereas the label intensity by SDS-FRL reflected the PtdIns(4,5)P2 amount. In PC12 cells, PtdIns(4,5)P2 was observed in a punctate pattern in the formaldehyde-fixed plasma membrane, whereas it was distributed randomly by SDS-FRL and showed clustering after formaldehyde fixation. The results indicate that the distribution of PtdIns(4,5)P2 can be defined most reliably by SDS-FRL of quick-frozen cells., (© 2024 Tsuji et al.)

For accurate mitosis, all chromosomes must achieve "biorientation," with replicated sister chromatids coupled via kinetochores to the plus ends of opposing microtubules. However, kinetochores first bind the sides of microtubules and subsequently find plus ends through a trial-and-error process; accurate biorientation depends on the selective release of erroneous attachments. Proposed mechanisms for error-correction have focused mainly on plus-end attachments. Whether erroneous side attachments are distinguished from correct side attachments is unknown. Here, we show that side-attached kinetochores are very sensitive to microtubule polarity, gripping sixfold more strongly when pulled toward plus versus minus ends. This directionally asymmetric grip is conserved in human and yeast subcomplexes, and it correlates with changes in the axial arrangement of subcomplexes within the kinetochore, suggesting that internal architecture dictates attachment strength. We propose that the kinetochore's directional grip promotes accuracy during early mitosis by stabilizing correct attachments even before both sisters have found plus ends., (© 2024 Larson et al.)

The mitochondria-ER-cortex anchor (MECA) forms a tripartite membrane contact site between mitochondria, the endoplasmic reticulum (ER), and the plasma membrane (PM). The core component of MECA, Num1, interacts with the PM and mitochondria via two distinct lipid-binding domains; however, the molecular mechanism by which Num1 interacts with the ER is unclear. Here, we demonstrate that Num1 contains a FFAT motif in its C-terminus that interacts with the integral ER membrane protein Scs2. While dispensable for Num1's functions in mitochondrial tethering and dynein anchoring, the FFAT motif is required for Num1's role in promoting mitochondrial division. Unexpectedly, we also reveal a novel function of MECA in regulating the distribution of phosphatidylinositol-4-phosphate (PI(4)P). Breaking Num1 association with any of the three membranes it tethers results in an accumulation of PI(4)P on the PM, likely via disrupting Sac1-mediated PI(4)P turnover. This work establishes MECA as an important regulatory hub that spatially organizes mitochondria, ER, and PM to coordinate crucial cellular functions., (© 2024 Casler et al.)

Autophagy is essential for maintaining glucose homeostasis. However, the mechanism by which cells sense and respond to glucose starvation to induce autophagy remains incomplete. Here, we show that calcium serves as a fundamental triggering signal that connects environmental sensing to the formation of the autophagy initiation complex during glucose starvation. Mechanistically, glucose starvation instigates the release of vacuolar calcium into the cytoplasm, thus triggering the activation of Rck2 kinase. In turn, Rck2-mediated Atg11 phosphorylation enhances Atg11 interactions with Bmh1/2 bound to the Snf1-Sip1-Snf4 complex, leading to recruitment of vacuolar membrane-localized Snf1 to the PAS and subsequent Atg1 activation, thereby initiating autophagy. We also identified Glc7, a protein phosphatase-1, as a critical regulator of the association between Bmh1/2 and the Snf1 complex. We thus propose that calcium-triggered Atg11-Bmh1/2-Snf1 complex assembly initiates autophagy by controlling Snf1-mediated Atg1 activation in response to glucose starvation., (© 2024 Yao et al.)

Eukaryotic ribosomal proteins contain extended regions essential for translation coordination. Dedicated chaperones stabilize the associated ribosomal proteins. We identified Bcp1 as the chaperone of uL14 in Saccharomyces cerevisiae. Rkm1, the lysine methyltransferase of uL14, forms a ternary complex with Bcp1 and uL14 to protect uL14. Rkm1 is transported with uL14 by importins to the nucleus, and Bcp1 disassembles Rkm1 and importin from uL14 simultaneously in a RanGTP-independent manner. Molecular docking, guided by crosslinking mass spectrometry and validated by a low-resolution cryo-EM map, reveals the correlation between Bcp1, Rkm1, and uL14, demonstrating the protection model. In addition, the ternary complex also serves as a surveillance point, whereas incorrect uL14 is retained on Rkm1 and prevented from loading to the pre-60S ribosomal subunits. This study reveals the molecular mechanism of how uL14 is protected and quality checked by serial steps to ensure its safe delivery from the cytoplasm until its incorporation into the 60S ribosomal subunit., (© 2024 Yeh et al.)

Cell growth is required for cell cycle progression. The amount of growth required for cell cycle progression is reduced in poor nutrients, which leads to a reduction in cell size. In budding yeast, nutrients can influence cell size by modulating the extent of bud growth, which occurs predominantly in mitosis. However, the mechanisms are unknown. Here, we used mass spectrometry to identify proteins that modulate bud growth in response to nutrient availability. This led to the discovery that nutrients regulate numerous components of the mitotic exit network (MEN), which controls exit from mitosis. A key component of the MEN undergoes gradual multisite phosphorylation during bud growth that is dependent upon bud growth and correlated with the extent of growth. Furthermore, activation of the MEN is sufficient to override a growth requirement for mitotic exit. The data suggest a model in which the MEN ensures that mitotic exit occurs only when an appropriate amount of bud growth has occurred., (© 2024 Talavera et al.)

Autophagy serves as a stress response pathway by mediating the degradation of cellular material within lysosomes. In autophagy, this material is encapsulated in double-membrane vesicles termed autophagosomes, which form from precursors referred to as phagophores. Phagophores grow by lipid influx from the endoplasmic reticulum into Atg9-positive compartments and local lipid synthesis provides lipids for their expansion. How phagophore nucleation and expansion are coordinated with lipid synthesis is unclear. Here, we show that Faa1, an enzyme activating fatty acids, is recruited to Atg9 vesicles by directly binding to negatively charged membranes with a preference for phosphoinositides such as PI3P and PI4P. We define the membrane-binding surface of Faa1 and show that its direct interaction with the membrane is required for its recruitment to phagophores. Furthermore, the physiological localization of Faa1 is key for its efficient catalysis and promotes phagophore expansion. Our results suggest a positive feedback loop coupling phagophore nucleation and expansion to lipid synthesis., (© 2024 Baumann et al.)

The essential Golgi protein Sly1 is a member of the Sec1/mammalian Unc-18 (SM) family of SNARE chaperones. Sly1 was originally identified through remarkable gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory but also acts as a positive effector. An amphipathic lipid packing sensor (ALPS)-like helix within the loop directly binds high-curvature membranes. Membrane binding is required for relief of Sly1 autoinhibition and also allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 mutation bypasses requirements for diverse tethering factors but loses this ability if the tethering activity is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to initiate trans-SNARE complex assembly and fusion in the early secretory pathway., (© 2024 Duan et al.)

SM proteins including Sly1 are essential cofactors of SNARE-mediated membrane fusion. Using SNARE and Sly1 mutants and chemically defined in vitro assays, we separate and assess proposed mechanisms through which Sly1 augments fusion: (i) opening the closed conformation of the Qa-SNARE Sed5; (ii) close-range tethering of vesicles to target organelles, mediated by the Sly1-specific regulatory loop; and (iii) nucleation of productive trans-SNARE complexes. We show that all three mechanisms are important and operate in parallel, and that close-range tethering promotes trans-complex assembly when cis-SNARE assembly is a competing process. Further, we demonstrate that the autoinhibitory N-terminal Habc domain of Sed5 has at least two positive activities: it is needed for correct Sed5 localization, and it directly promotes Sly1-dependent fusion. "Split Sed5," with Habc presented solely as a soluble fragment, can function both in vitro and in vivo. Habc appears to facilitate events leading to lipid mixing rather than promoting opening or stability of the fusion pore., (© 2024 Duan et al.)

Organelles of the endomembrane system contain Rab GTPases as identity markers. Their localization is determined by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). It remains largely unclear how these regulators are specifically targeted to organelles and how their activity is regulated. Here, we focus on the GAP Gyp7, which acts on the Rab7-like Ypt7 protein in yeast, and surprisingly observe the protein exclusively in puncta proximal to the vacuole. Mistargeting of Gyp7 to the vacuole strongly affects vacuole morphology, suggesting that endosomal localization is needed for function. In agreement, efficient endolysosomal transport requires Gyp7. In vitro assays reveal that Gyp7 requires a distinct lipid environment for membrane binding and activity. Overexpression of Gyp7 concentrates Ypt7 in late endosomes and results in resistance to rapamycin, an inhibitor of the target of rapamycin complex 1 (TORC1), suggesting that these late endosomes are signaling endosomes. We postulate that Gyp7 is part of regulatory machinery involved in late endosome function., (© 2024 Füllbrunn et al.)

The septin cytoskeleton is extensively regulated by posttranslational modifications, such as phosphorylation, to achieve the diversity of architectures including rings, hourglasses, and gauzes. While many of the phosphorylation events of septins have been extensively studied in the budding yeast Saccharomyces cerevisiae, the regulation of the kinases involved remains poorly understood. Here, we show that two septin-associated kinases, the LKB1/PAR-4-related kinase Elm1 and the Nim1/PAR-1-related kinase Gin4, regulate each other at two discrete points of the cell cycle. During bud emergence, Gin4 targets Elm1 to the bud neck via direct binding and phosphorylation to control septin hourglass assembly and stability. During mitosis, Elm1 maintains Gin4 localization via direct binding and phosphorylation to enable timely remodeling of the septin hourglass into a double ring. This mutual control between Gin4 and Elm1 ensures that septin architecture is assembled and remodeled in a temporally controlled manner to perform distinct functions during the cell cycle., (© 2024 Marquardt et al.)

The highly conserved HEATR5 proteins are best known for their roles in membrane traffic mediated by the adaptor protein complex-1 (AP1). HEATR5 proteins rely on fast-evolving cofactors to bind to AP1. However, how HEATR5 proteins interact with these cofactors is unknown. Here, we report that the budding yeast HEATR5 protein, Laa1, functions in two biochemically distinct complexes. These complexes are defined by a pair of mutually exclusive Laa1-binding proteins, Laa2 and the previously uncharacterized Lft1/Yml037c. Despite limited sequence similarity, biochemical analysis and structure predictions indicate that Lft1 and Laa2 bind Laa1 via structurally similar mechanisms. Both Laa1 complexes function in intra-Golgi recycling. However, only the Laa2-Laa1 complex binds to AP1 and contributes to its localization. Finally, structure predictions indicate that human HEATR5 proteins bind to a pair of fast-evolving interacting partners via a mechanism similar to that observed in yeast. These results reveal mechanistic insight into how HEATR5 proteins bind their cofactors and indicate that Laa1 performs functions besides recruiting AP1., (© 2024 Marmorale et al.)

Cells store lipids in the form of triglyceride (TG) and sterol ester (SE) in lipid droplets (LDs). Distinct pools of LDs exist, but a pervasive question is how proteins localize to and convey functions to LD subsets. Here, we show that the yeast protein YDR275W/Tld1 (for TG-associated LD protein 1) localizes to a subset of TG-containing LDs and reveal it negatively regulates lipolysis. Mechanistically, Tld1 LD targeting requires TG, and it is mediated by two distinct hydrophobic regions (HRs). Molecular dynamics simulations reveal that Tld1's HRs interact with TG on LDs and adopt specific conformations on TG-rich LDs versus SE-rich LDs in yeast and human cells. Tld1-deficient yeast display no defect in LD biogenesis but exhibit elevated TG lipolysis dependent on lipase Tgl3. Remarkably, overexpression of Tld1, but not LD protein Pln1/Pet10, promotes TG accumulation without altering SE pools. Finally, we find that Tld1-deficient cells display altered LD mobilization during extended yeast starvation. We propose that Tld1 senses TG-rich LDs and regulates lipolysis on LD subpopulations., (© 2023 Speer et al.)

Understanding how numerous actin-binding proteins (ABPs) work in concert to control the assembly, organization, and turnover of the actin cytoskeleton requires quantitative information about the levels of each component. Here, we measured the cellular concentrations of actin and the majority of the conserved ABPs in Saccharomyces cerevisiae, as well as the free (cytosolic) fractions of each ABP. The cellular concentration of actin is estimated to be 13.2 µM, with approximately two-thirds in the F-actin form and one-third in the G-actin form. Cellular concentrations of ABPs range from 12.4 to 0.85 µM (Tpm1> Pfy1> Cof1> Abp1> Srv2> Abp140> Tpm2> Aip1> Cap1/2> Crn1> Sac6> Twf1> Arp2/3> Scp1). The cytosolic fractions of all ABPs are unexpectedly high (0.6-0.9) and remain so throughout the cell cycle. Based on these numbers, we speculate that F-actin binding sites are limited in vivo, which leads to high cytosolic levels of ABPs, and in turn helps drive the rapid assembly and turnover of cellular F-actin structures., (© 2023 Gonzalez Rodriguez et al.)

Mitochondria are highly dynamic double membrane-bound organelles that maintain their shape in part through fission and fusion. Mitochondrial fission is performed by a dynamin-related protein, Dnm1 (Drp1 in humans), that constricts and divides the mitochondria in a GTP hydrolysis-dependent manner. However, it is unclear whether factors inside mitochondria help coordinate the process and if Dnm1/Drp1 activity is sufficient to complete the fission of both mitochondrial membranes. Here, we identify an intermembrane space protein required for mitochondrial fission in yeast, which we propose to name Mdi1 (also named Atg44). Loss of Mdi1 causes mitochondrial hyperfusion due to defects in fission, but not the lack of Dnm1 recruitment to mitochondria. Mdi1 is conserved in fungal species, and its homologs contain an amphipathic α-helix, mutations of which disrupt mitochondrial morphology. One model is that Mdi1 distorts mitochondrial membranes to enable Dnm1 to robustly complete fission. Our work reveals that Dnm1 cannot efficiently divide mitochondria without the coordinated function of Mdi1 inside mitochondria., (© 2023 Connor et al.)

Proteins with multiple membrane-spanning segments (MS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. Shr3, an ER membrane-localized chaperone in Saccharomyces cerevisiae, is required for the functional expression of a family of 18 amino acid permeases (AAP) comprised of 12 MS. We have used comprehensive scanning mutagenesis and deletion analysis of Shr3 combined with a modified split-ubiquitin approach to probe chaperone-substrate interactions in vivo. Shr3 selectively interacts with nested C-terminal AAP truncations in marked contrast to similar truncations of non-Shr3 substrate sugar transporters. Shr3-AAP interactions initiate with the first four MS of AAP and successively strengthen but weaken abruptly when all 12 MS are present. Shr3-AAP interactions are based on structural rather than sequence-specific interactions involving membrane and luminal domains of Shr3. The data align with Shr3 engaging nascent N-terminal chains of AAP, functioning as a scaffold to facilitate folding as translation completes., (© 2023 Myronidi et al.)

The nuclear pore complex (NPC) physically interacts with chromatin and regulates gene expression. The Saccharomyces cerevisiae inner ring nucleoporin Nup170 has been implicated in chromatin organization and the maintenance of gene silencing in subtelomeric regions. To gain insight into how Nup170 regulates this process, we used protein-protein interactions, genetic interactions, and transcriptome correlation analyses to identify the Ctf18-RFC complex, an alternative proliferating cell nuclear antigen (PCNA) loader, as a facilitator of the gene regulatory functions of Nup170. The Ctf18-RFC complex is recruited to a subpopulation of NPCs that lack the nuclear basket proteins Mlp1 and Mlp2. In the absence of Nup170, PCNA levels on DNA are reduced, resulting in the loss of silencing of subtelomeric genes. Increasing PCNA levels on DNA by removing Elg1, which is required for PCNA unloading, rescues subtelomeric silencing defects in nup170Δ. The NPC, therefore, mediates subtelomeric gene silencing by regulating PCNA levels on DNA., (© 2023 Kumar et al.)

In macroautophagy, cellular components are sequestered within autophagosomes and transported to lysosomes/vacuoles for degradation. Although phosphatidylinositol 3-kinase complex I (PI3KCI) plays a pivotal role in the regulation of autophagosome biogenesis, little is known about how this complex localizes to the pre-autophagosomal structure (PAS). In Saccharomyces cerevisiae, PI3KCI is composed of PI3K Vps34 and conserved subunits Vps15, Vps30, Atg14, and Atg38. In this study, we discover that PI3KCI interacts with the vacuolar membrane anchor Vac8, the PAS scaffold Atg1 complex, and the pre-autophagosomal vesicle component Atg9 via the Atg14 C-terminal region, the Atg38 C-terminal region, and the Vps30 BARA domain, respectively. While the Atg14-Vac8 interaction is constitutive, the Atg38-Atg1 complex interaction and the Vps30-Atg9 interaction are enhanced upon macroautophagy induction depending on Atg1 kinase activity. These interactions cooperate to target PI3KCI to the PAS. These findings provide a molecular basis for PAS targeting of PI3KCI during autophagosome biogenesis., (© 2023 Hitomi et al.)

As eukaryotic cells progress through cell division, the nuclear envelope (NE) membrane must expand to accommodate the formation of progeny nuclei. In Saccharomyces cerevisiae, closed mitosis allows visualization of NE biogenesis during mitosis. During this period, the SUMO E3 ligase Siz2 binds the inner nuclear membrane (INM) and initiates a wave of INM protein SUMOylation. Here, we show these events increase INM levels of phosphatidic acid (PA), an intermediate of phospholipid biogenesis, and are necessary for normal mitotic NE membrane expansion. The increase in INM PA is driven by the Siz2-mediated inhibition of the PA phosphatase Pah1. During mitosis, this results from the binding of Siz2 to the INM and dissociation of Spo7 and Nem1, a complex required for the activation of Pah1. As cells enter interphase, the process is then reversed by the deSUMOylase Ulp1. This work further establishes a central role for temporally controlled INM SUMOylation in coordinating processes, including membrane expansion, that regulate NE biogenesis during mitosis., (© 2023 Saik et al.)

The nuclear envelope (NE) is important in maintaining genome organization. The role of lipids in communication between the NE and telomere regulation was investigated, including how changes in lipid composition impact gene expression and overall nuclear architecture. Yeast was treated with the non-metabolizable lysophosphatidylcholine analog edelfosine, known to accumulate at the perinuclear ER. Edelfosine induced NE deformation and disrupted telomere clustering but not anchoring. Additionally, the association of Sir4 at telomeres decreased. RNA-seq analysis showed altered expression of Sir-dependent genes located at sub-telomeric (0-10 kb) regions, consistent with Sir4 dispersion. Transcriptomic analysis revealed that two lipid metabolic circuits were activated in response to edelfosine, one mediated by the membrane sensing transcription factors, Spt23/Mga2, and the other by a transcriptional repressor, Opi1. Activation of these transcriptional programs resulted in higher levels of unsaturated fatty acids and the formation of nuclear lipid droplets. Interestingly, cells lacking Sir proteins displayed resistance to unsaturated-fatty acids and edelfosine, and this phenotype was connected to Rap1., (© 2023 Sosa Ponce et al.)

During autophagy, rapid membrane assembly expands small phagophores into large double-membrane autophagosomes. Theoretical modeling predicts that the majority of autophagosomal phospholipids are derived from highly efficient non-vesicular phospholipid transfer (PLT) across phagophore-ER contacts (PERCS). Currently, the phagophore-ER tether Atg2 is the only PLT protein known to drive phagophore expansion in vivo. Here, our quantitative live-cell imaging analysis reveals a poor correlation between the duration and size of forming autophagosomes and the number of Atg2 molecules at PERCS of starving yeast cells. Strikingly, we find that Atg2-mediated PLT is non-rate limiting for autophagosome biogenesis because membrane tether and the PLT protein Vps13 localizes to the rim and promotes the expansion of phagophores in parallel with Atg2. In the absence of Vps13, the number of Atg2 molecules at PERCS determines the duration and size of forming autophagosomes with an apparent in vivo transfer rate of ∼200 phospholipids per Atg2 molecule and second. We propose that conserved PLT proteins cooperate in channeling phospholipids across organelle contact sites for non-rate-limiting membrane assembly during autophagosome biogenesis., (© 2023 Dabrowski et al.)

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and built from ∼30 different nucleoporins (Nups) in multiple copies, few are integral membrane proteins. One of these transmembrane nucleoporins, Ndc1, is thought to function in NPC assembly at the fused inner and outer nuclear membranes. Here, we show a direct interaction of Ndc1's transmembrane domain with Nup120 and Nup133, members of the pore membrane coating Y-complex. We identify an amphipathic helix in Ndc1's C-terminal domain binding highly curved liposomes. Upon overexpression, this amphipathic motif is toxic and dramatically alters the intracellular membrane organization in yeast. Ndc1's amphipathic motif functionally interacts with related motifs in the C-terminus of the nucleoporins Nup53 and Nup59, important for pore membrane binding and interconnecting NPC modules. The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53. Our data indicate that nuclear membrane and presumably NPC biogenesis depends on a balanced ratio between amphipathic motifs in diverse nucleoporins., (© 2023 Amm et al.)

Ceramides are essential precursors of complex sphingolipids and act as potent signaling molecules. Ceramides are synthesized in the endoplasmic reticulum (ER) and receive their head-groups in the Golgi apparatus, yielding complex sphingolipids (SPs). Transport of ceramides between the ER and the Golgi is executed by the essential ceramide transport protein (CERT) in mammalian cells. However, yeast cells lack a CERT homolog, and the mechanism of ER to Golgi ceramide transport remains largely elusive. Here, we identified a role for yeast Svf1 in ceramide transport between the ER and the Golgi. Svf1 is dynamically targeted to membranes via an N-terminal amphipathic helix (AH). Svf1 binds ceramide via a hydrophobic binding pocket that is located in between two lipocalin domains. We showed that Svf1 membrane-targeting is important to maintain flux of ceramides into complex SPs. Together, our results show that Svf1 is a ceramide binding protein that contributes to sphingolipid metabolism at Golgi compartments., (© 2023 Limar et al.)

The essential COPI coat mediates retrieval of transmembrane proteins at the Golgi and endosomes following recruitment by the small GTPase, Arf1. ArfGAP proteins regulate COPI coats, but molecular details for COPI recognition by ArfGAPs remain elusive. Biochemical and biophysical data reveal how β'-COP propeller domains directly engage the yeast ArfGAP, Glo3, with a low micromolar binding affinity. Calorimetry data demonstrate that both β'-COP propeller domains are required to bind Glo3. An acidic patch on β'-COP (D437/D450) interacts with Glo3 lysine residues located within the BoCCS (binding of coatomer, cargo, and SNAREs) region. Targeted point mutations in either Glo3 BoCCS or β'-COP abrogate the interaction in vitro, and loss of the β'-COP/Glo3 interaction drives Ste2 missorting to the vacuole and aberrant Golgi morphology in budding yeast. These data suggest that cells require the β'-COP/Glo3 interaction for cargo recycling via endosomes and the TGN, where β'-COP serves as a molecular platform to coordinate binding to multiple proteins, including Glo3, Arf1, and the COPI F-subcomplex., (© 2023 Xie et al.)

How cells simultaneously assemble actin structures of distinct sizes, shapes, and filamentous architectures is still not well understood. Here, we used budding yeast as a model to investigate how competition for the barbed ends of actin filaments might influence this process. We found that while vertebrate capping protein (CapZ) and formins can simultaneously associate with barbed ends and catalyze each other's displacement, yeast capping protein (Cap1/2) poorly displaces both yeast and vertebrate formins. Consistent with these biochemical differences, in vivo formin-mediated actin cable assembly was strongly attenuated by the overexpression of CapZ but not Cap1/2. Multiwavelength live cell imaging further revealed that actin patches in cap2∆ cells acquire cable-like features over time, including recruitment of formins and tropomyosin. Together, our results suggest that the activities of S. cerevisiae Cap1/2 have been tuned across evolution to allow robust cable assembly by formins in the presence of high cytosolic levels of Cap1/2, which conversely limit patch growth and shield patches from formins., (© 2023 Wirshing et al.)

How cells regulate α- and β-tubulin to meet the demand for αβ-heterodimers and avoid consequences of monomer imbalance is not understood. We investigate the role of gene copy number and how shifting expression of α- or β-tubulin genes impacts tubulin proteostasis and microtubule function in Saccharomyces cerevisiae. We find that α-tubulin gene copy number is important for maintaining excess α-tubulin protein compared to β-tubulin protein. Excess α-tubulin prevents accumulation of super-stoichiometric β-tubulin, which leads to loss of microtubules, formation of non-microtubule assemblies of tubulin, and disrupts cell proliferation. In contrast, sub-stoichiometric β-tubulin or overexpression of α-tubulin has minor effects. We provide evidence that yeast cells equilibrate α-tubulin protein concentration when α-tubulin isotype expression is increased. We propose an asymmetric relationship between α- and β-tubulins, in which α-tubulins are maintained in excess to supply αβ-heterodimers and limit the accumulation of β-tubulin monomers., (© 2023 Wethekam and Moore.)

The exocyst imparts spatial control during exocytic vesicle tethering through its interactions with proteins and lipids on the vesicle and the plasma membrane. One such interaction is with the vesicle tether Sro7, although the outcome of this interaction is poorly understood. Here, we describe how Sro7 binding to the Exo84 subunit results in activation of the exocyst complex which leads to an increase in avidity for the Rab GTPase Sec4 and an increase in exocyst-mediated vesicle tethering. Gain-of-function (GOF) mutations in Exo84 that mimic Sro7 activation replicate these biochemical changes and result in allosteric changes within the complex. Direct comparison of GOF mutants which mimic Sro7- and Rho/Cdc42-activation of the exocyst reveals distinct mechanisms and outcomes. We propose a model by which these two activation pathways reside within the same tethering complex but remain insulated from one another. Structural modeling suggests a related mechanism for Sro7 activation of the exocyst in yeast and Ral GTPase activation of the exocyst in animal cells., (© 2023 Miller et al.)

Septins are an evolutionarily conserved protein family whose members form hetero-oligomeric complexes that assemble into filaments and higher-order structures. In this issue, Martins et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202203016) and Cannon et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202204063) report that heteromer composition impacts the physiological role of septin filaments in yeast and human cells., (© 2023 Güler and Mostowy.)

The mating of budding yeast depends on chemotropism, a fundamental cellular process. Haploid yeast cells of opposite mating type signal their positions to one another through mating pheromones. We have proposed a deterministic gradient sensing model that explains how these cells orient toward their mating partners. Using the cell-cycle determined default polarity site (DS), cells assemble a gradient tracking machine (GTM) composed of signaling, polarity, and trafficking proteins. After assembly, the GTM redistributes up the gradient, aligns with the pheromone source, and triggers polarized growth toward the partner. Since positive feedback mechanisms drive polarized growth at the DS, it is unclear how the GTM is released for tracking. What prevents the GTM from triggering polarized growth at the DS? Here, we describe two mechanisms that are essential for tracking: inactivation of the Ras GTPase Bud1 and positioning of actin-independent vesicle delivery upgradient., (© 2022 Wang et al.)

Rho GTPases are central regulators of cell polarity and signaling. How Rho GTPases are directed to function in certain settings remains unclear. Here, we show the protein levels of the yeast Rho GTPase Cdc42p are regulated, which impacts a subset of its biological functions. Specifically, the active conformation of Cdc42p was ubiquitinated by the NEDD4 ubiquitin ligase Rsp5p and HSP40/HSP70 chaperones and turned over in the proteasome. A GTP-locked (Q61L) turnover-defective (TD) version, Cdc42pQ61L+TD, hyperactivated the MAPK pathway that regulates filamentous growth (fMAPK). Cdc42pQ61L+TD did not influence the activity of the mating pathway, which shares components with the fMAPK pathway. The fMAPK pathway adaptor, Bem4p, stabilized Cdc42p levels, which resulted in elevated fMAPK pathway signaling. Our results identify Cdc42p turnover regulation as being critical for the regulation of a MAPK pathway. The control of Rho GTPase levels by stabilization and turnover may be a general feature of signaling pathway regulation, which can result in the execution of a specific developmental program., (© 2022 Gonzalez and Cullen.)

Upon internalization, many surface membrane proteins are recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labor between interconnected pathways are poorly defined. In yeast, we show recycling back to the surface occurs through distinct pathways. In addition to retrograde recycling pathways via the late Golgi, used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalize to, and recycle from, endosomes marked by the ESCRT-III associated factor Ist1. This compartment serves as both "early" and "recycling" endosome. We show Ist1 is ubiquitinated and that this is required for proper endosomal recruitment and cargo recycling to the surface. Additionally, the essential ATPase Cdc48 and its adaptor Npl4 are required for recycling, potentially through regulation of ubiquitinated Ist1. This collectively suggests mechanistic features of recycling from endosomes to the plasma membrane are conserved., (© 2022 Laidlaw et al.)

The chaperone-mediated sequestration of misfolded proteins into inclusions is a pivotal cellular strategy to maintain proteostasis in Saccharomyces cerevisiae, executed by small heat shock proteins (sHsps) Hsp42 and Btn2. Direct homologs of Hsp42 and Btn2 are absent in other organisms, questioning whether sequestration represents a conserved proteostasis strategy and, if so, which factors are involved. We examined sHsps from Escherchia coli, Caenorhabditis elegans, and humans for their ability to complement the defects of yeast sequestrase mutants. We show that sequestration of misfolded proteins is an original and widespread activity among sHsps executed by specific family members. Sequestrase positive C. elegans' sHsps harbor specific sequence features, including a high content of aromatic and methionine residues in disordered N-terminal extensions. Those sHsps buffer limitations in Hsp70 capacity in C. elegans WT animals and are upregulated in long-lived daf-2 mutants, contributing to lifespan extension. Cellular protection by sequestration of misfolded proteins is, therefore, an evolutionarily conserved activity of the sHsp family., (© 2022 Shrivastava et al.)

Endocytosis is a multistep process involving the sequential recruitment and action of numerous proteins. This process can be divided into two phases: an early phase, in which sites of endocytosis are formed, and a late phase in which clathrin-coated vesicles are formed and internalized into the cytosol, but how these phases link to each other remains unclear. In this study, we demonstrate that anchoring the yeast Eps15-like protein Pan1p to the peroxisome triggers most of the events occurring during the late phase at the peroxisome. At this ectopic location, Pan1p recruits most proteins that function in the late phases-including actin nucleation promoting factors-and then initiates actin polymerization. Pan1p also recruited Prk1 kinase and actin depolymerizing factors, thereby triggering disassembly immediately after actin assembly and inducing dissociation of endocytic proteins from the peroxisome. These observations suggest that Pan1p is a key regulator for initiating, processing, and completing the late phase of endocytosis., (© 2022 Enshoji et al.)

Lipid droplets (LDs) are essential for cellular lipid homeostasis by storing diverse neutral lipids (NLs), such as triacylglycerol (TAG), steryl esters (SE), and retinyl esters (RE). A proper assembly of TAG-containing LDs at the ER requires Seipin, a conserved protein often mutated in lipodystrophies. Here, we show that the yeast Seipin Sei1 and its partner Ldb16 also promote the storage of other NL in LDs. Importantly, this role of Sei1/Ldb16 is evolutionarily conserved as expression of human-Seipin restored normal SE-containing LDs in yeast Seipin mutants. As in the case of TAG, the formation of SE-containing LDs requires interactions between hydroxyl-residues in human Seipin or yeast Ldb16 with NL carboxyl esters. These findings provide a universal mechanism for Seipin-mediated LD formation and suggest a model for how Seipin distinguishes NLs from aliphatic phospholipid acyl chains in the center of the membrane bilayer., (© 2022 Renne et al.)

Membrane contact sites are specialized platforms formed between most organelles that enable them to exchange metabolites and influence the dynamics of each other. The yeast vacuole is a degradative organelle equivalent to the lysosome in higher eukaryotes with important roles in ion homeostasis and metabolism. Using a high-content microscopy screen, we identified Ymr160w (Cvm1, for contact of the vacuole membrane 1) as a novel component of three different contact sites of the vacuole: with the nuclear endoplasmic reticulum, the mitochondria, and the peroxisomes. At the vacuole-mitochondria contact site, Cvm1 acts as a tether independently of previously known tethers. We show that changes in Cvm1 levels affect sphingolipid homeostasis, altering the levels of multiple sphingolipid classes and the response of sphingolipid-sensing signaling pathways. Furthermore, the contact sites formed by Cvm1 are induced upon a decrease in sphingolipid levels. Altogether, our work identifies a novel protein that forms multiple contact sites and supports a role of lysosomal contacts in sphingolipid homeostasis., (© 2022 Bisinski et al.)

Tail-anchored (TA) membrane proteins have a potential risk to be mistargeted to the mitochondrial outer membrane (OM). Such mislocalized TA proteins can be extracted by the mitochondrial AAA-ATPase Msp1 from the OM and transferred to the ER for ER protein quality control involving ubiquitination by the ER-resident Doa10 complex. Yet it remains unclear how the extracted TA proteins can move to the ER crossing the aqueous cytosol and whether this transfer to the ER is essential for the clearance of mislocalized TA proteins. Here we show by time-lapse microscopy that mislocalized TA proteins, including an authentic ER-TA protein, indeed move from mitochondria to the ER in a manner strictly dependent on Msp1 expression. The Msp1-dependent mitochondria-to-ER transfer of TA proteins is blocked by defects in the GET system, and this block is not due to impaired Doa10 functions. Thus, the GET pathway facilitates the transfer of mislocalized TA proteins from mitochondria to the ER., (© 2022 Matsumoto et al.)

The endomembrane system of eukaryotic cells is essential for cellular homeostasis during growth and proliferation. Previous work showed that a central regulator of growth, namely the target of rapamycin complex 1 (TORC1), binds both membranes of vacuoles and signaling endosomes (SEs) that are distinct from multivesicular bodies (MVBs). Interestingly, the endosomal TORC1, which binds membranes in part via the EGO complex, critically defines vacuole integrity. Here, we demonstrate that SEs form at a branch point of the biosynthetic and endocytic pathways toward the vacuole and depend on MVB biogenesis. Importantly, function of the HOPS tethering complex is essential to maintain the identity of SEs and proper endosomal and vacuolar TORC1 activities. In HOPS mutants, the EGO complex redistributed to the Golgi, which resulted in a partial mislocalization of TORC1. Our study uncovers that SE function requires a functional HOPS complex and MVBs, suggesting a tight link between trafficking and signaling along the endolysosomal pathway., (© 2022 Gao et al.)

The mitochondrial outer membrane (MOM) harbors proteins that traverse the membrane via several helical segments and are called multi-span proteins. To obtain new insights into the biogenesis of these proteins, we utilized yeast mitochondria and the multi-span protein Om14. Testing different truncation variants, we show that while only the full-length protein contains all the information that assures perfect targeting specificity, shorter variants are targeted to mitochondria with compromised fidelity. Employing a specific insertion assay and various deletion strains, we show that proteins exposed to the cytosol do not contribute significantly to the biogenesis process. We further demonstrate that Mim1 and Porin support optimal membrane integration of Om14 but none of them are absolutely required. Unfolding of newly synthesized Om14, its optimal hydrophobicity, and higher fluidity of the membrane enhanced the import capacity of Om14. Collectively, these findings suggest that MOM multi-span proteins follow different biogenesis pathways in which proteinaceous elements and membrane behavior contribute to a variable extent to the combined efficiency., (© 2022 Zhou et al.)

The lysosome is the major catabolic organelle in the cell that has been established as a key metabolic signaling center. Mutations in many lysosomal proteins have catastrophic effects and cause neurodegeneration, cancer, and age-related diseases. The vacuole is the lysosomal analog of Saccharomyces cerevisiae that harbors many evolutionary conserved proteins. Proteins reach vacuoles via the Vps10-dependent endosomal vacuolar protein sorting pathway, via the alkaline phosphatase (ALP or AP-3) pathway, and via the cytosol-to-vacuole transport (CVT) pathway. A systematic understanding of the cargo spectrum of each pathway is completely lacking. Here, we use quantitative proteomics of purified vacuoles to generate the yeast lysosomal biogenesis map. This dataset harbors information on the cargo-receptor relationship of almost all vacuolar proteins. We map binding motifs of Vps10 and the AP-3 complex and identify a novel cargo of the CVT pathway under nutrient-rich conditions. Our data show how organelle purification and quantitative proteomics can uncover fundamental insights into organelle biogenesis., (© 2022 Eising et al.)

Acidification of secretory and endocytic organelles is required for proper receptor recycling, membrane traffic, protein degradation, and solute transport. Proton-pumping vacuolar H+ ATPases (V-ATPases) are responsible for this luminal acidification, which increases progressively as secretory and endocytic vesicles mature. An increasing density of V-ATPase complexes is thought to account for the gradual decrease in pH, but available reagents have not been sufficiently sensitive or specific to test this hypothesis. We introduce a new probe to localize and quantify V-ATPases. The probe is derived from SidK, a Legionella pneumophila effector protein that binds to the V-ATPase A subunit. We generated plasmids encoding fluorescent chimeras of SidK1-278, and labeled recombinant SidK1-278 with Alexa Fluor 568 to visualize and quantify V-ATPases with high specificity in live and fixed cells, respectively. We show that V-ATPases are acquired progressively during phagosome maturation, that they distribute in discrete membrane subdomains, and that their density in lysosomes depends on their subcellular localization., (© 2022 Maxson et al.)

Glycosylphosphatidylinositol (GPI) is a glycolipid membrane anchor found on surface proteins in all eukaryotes. It is synthesized in the ER membrane. Each GPI anchor requires three molecules of ethanolamine phosphate (P-Etn), which are derived from phosphatidylethanolamine (PE). We found that efficient GPI anchor synthesis in Saccharomyces cerevisiae requires Csf1; cells lacking Csf1 accumulate GPI precursors lacking P-Etn. Structure predictions suggest Csf1 is a tube-forming lipid transport protein like Vps13. Csf1 is found at contact sites between the ER and other organelles. It interacts with the ER protein Mcd4, an enzyme that adds P-Etn to nascent GPI anchors, suggesting Csf1 channels PE to Mcd4 in the ER at contact sites to support GPI anchor biosynthesis. CSF1 has orthologues in Caenorhabditis elegans (lpd-3) and humans (KIAA1109/TWEEK); mutations in KIAA1109 cause the autosomal recessive neurodevelopmental disorder Alkuraya-Kučinskas syndrome. Knockout of lpd-3 and knockdown of KIAA1109 reduced GPI-anchored proteins on the surface of cells, suggesting Csf1 orthologues in human cells support GPI anchor biosynthesis., (© 2022 Toulmay et al.)

Arsenic is an environmental toxin that exists mainly as pentavalent arsenate and trivalent arsenite. Both forms activate the yeast SAPK Hog1 but with different consequences. We describe a mechanism by which cells distinguish between these arsenicals through one-step metabolism to differentially regulate the bidirectional glycerol channel Fps1, an adventitious port for arsenite. Cells exposed to arsenate reduce it to thiol-reactive arsenite, which modifies a set of cysteine residues in target proteins, whereas cells exposed to arsenite metabolize it to methylarsenite, which modifies an additional set of cysteine residues. Hog1 becomes arsenylated, which prevents it from closing Fps1. However, this block is overcome in cells exposed to arsenite through methylarsenylation of Acr3, an arsenite efflux pump that we found also regulates Fps1 directly. This adaptation allows cells to restrict arsenite entry through Fps1 and also allows its exit when produced from arsenate exposure. These results have broad implications for understanding how SAPKs activated by diverse stressors can drive stress-specific outputs., (© 2022 Lee and Levin.)