Biology of the Cell

Endosomal sorting complex required for transport (ESCRT-III) is a membrane remodeling complex involved in a large number of cellular processes. It appears to perform an essential function in eukaryotes, since to date no eukaryotic organism completely devoid of ESCRT-III has been found. Yet, yeast cells with a deletion of all eight known ESCRT-III genes are viable. We therefore searched for new, previously undiscovered ESCRT-III like proteins in yeast. HHPred uncovered several proteins with similarity to Snf7. The similarity was mostly restricted to the 1-2 hairpin region of Snf7. A conserved pattern of amino acids was detected in this region. The protein encoded by ORF YPL199c strikingly resembled Snf7 in its secondary structure. Since this protein could be the ninth member of the ESCRT-III family in yeast, we called it Nbr9 ("number nine"). Nbr9 is palmitoylated and localizes to the plasma membrane. In contrast to other palmitoylated proteins, it is not associated with lipid rafts. When NBR9 was deleted in the octuple ESCRT-III deletion background, the yeast cells were still viable. However, despite a number of experiments, we do not have evidence at present that Nbr9 is part of an alternative ESCRT-III complex.

FRET is a powerful tool to simultaneously establish and localize interactions between fluorescently tagged proteins with high spatial resolution. Rainey K.H. and Patterson G.H. introduced Photoswitching FRET (psFRET) using the Dronpa Photoswitching fluorescent protein. We present a straightforward detailed method, and a powerful software tool that allows adaptation of psFRET to diverse experimental setups. Image stacks, recording the decay of the Dronpa donor, serve as input to the software utility that includes effective preprocessing options preceding the calculation FRET efficiency at the single pixel level. We applied psFRET to generate interaction maps analyzing diverse interactions between cargo proteins, the GTPase Rab1b, and GRASP65 during ER to Golgi trafficking. Cargo-Rab1b interactions were restricted to the transit period from ER to Golgi. These data lend support to a mechanism whereby cargo sensing may regulate the level of downstream effectors recruitment to secretory membranes by Rab1.

Kinesin-3 KIF1A (UNC-104 in C. elegans) is the major axonal transporter of synaptic vesicles and mutations in this molecular motor are linked to KIF1A-associated neurological disorders (KAND) including Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis and hereditary spastic paraplegia. UNC-104 binds via its PH (pleckstrin homology) domain to the lipid bilayers of membranous vesicles which is considered a weak interaction. RT-PCR and Western blot experiments reveal genetic relations between SYD-2, UNC-10 and RAB-3. Co-immunoprecipitation assays reveal functional relations and bimolecular fluorescence complementation (BiFC) assays expose in situ interactions between these proteins. Though both SNB-1 and RAB-3 are actively transported by UNC-104, the movement of RAB-3 is generally enhanced and largely depending on the presence of SYD-2/UNC-10. Deletion of UNC-104s PH domain did not affect UNC-104/RAB-3 colocalization but did affect UNC-104/SNB-1 colocalization. Similarly, motility of RAB-3-labeled vesicles is unaltered in nematodes carrying a point mutation in the PH domain while movement of SNB-1 is significantly reduced in anterograde directions. These findings suggest a dual UNC-10/SYD-2 linker acting as a sufficient buttress to connect the motor to RAB-3-containing vesicles to enhance their transport. This additional linker will also strengthen the rather weak motor-lipid interaction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/723247v4_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@5e41c0org.highwire.dtl.DTLVardef@2ed9c8org.highwire.dtl.DTLVardef@1dbdb9dorg.highwire.dtl.DTLVardef@12f30c2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pex23 family proteins are membrane proteins of the endoplasmic reticulum that play a role in peroxisome and lipid body formation. The yeast Hansenula polymorpha contains four members: Pex23, Pex24, Pex29 and Pex32. We previously showed that the loss of Pex24 or Pex32 results in severe peroxisomal defects, caused by reduced peroxisome-endoplasmic reticulum membrane contact sites. We now analyzed whether the absence of Pex23 proteins affects other organelles. Vacuoles were normal in all deletion strains. The number of lipid droplets was reduced in pex23 and pex29, but not in pex24 and pex32, indicating that peroxisome and lipid droplet formation require different Pex23 proteins. In pex23 and pex29 cells, mitochondria were fragmented and clustered. This phenotype was not suppressed by an artificial mitochondria-endoplasmic reticulum tether, indicating that the abnormalities were not caused by reduced membrane contact sites. Deletion of DNM1 in pex23 cells partially suppressed the phenotype. Also, the level of the mitochondrial fusion protein Fzo1 was reduced in pex23 and pex29 cells. These observations indicate that certain Pex23 family proteins are required for normal mitochondrial fusion.

Auxin-inducible degradation is an increasingly popular protein-targeting reverse genetics approach. Use of this method has revolutionized the types of questions cell and molecular biologists can answer, however, a growing number of studies point to auxin alone impacting different cellular phenotypes. This study investigated the impact of different medium and auxin combinations on Saccharomyces cerevisiae growth. We observed that both natural and synthetic auxin (Indole-3-acetic acid (IAA) and 1-Naphthaleneacetic acid (NAA) respectively) impaired budding yeast growth in nutrient minimal but not nutrient rich media. This finding was true across different yeast strains with or without an intact auxin-inducible degradation system. Ultimately, this study highlights the need for proper controls when using auxin-inducible degradation.

The Endoplasmic Reticulum (ER) is the entry site to the secretory pathway, serving as the targeting destination for [~]30% of the proteome. The mechanisms for targeting soluble or integral membrane secretory pathway proteins are well-studied. However, it is currently unknown how the tens of ER surface proteins (SuPs), central for organelle function, reach the outer leaflet of the membrane. It was previously shown that an amphipathic helix (AH) from the Brome mosaic virus protein 1a, is both necessary and sufficient for targeting to the ER surface in bakers yeast. We therefore utilized this helix as a model substrate and performed a high-content screen to uncover factors that affect SuP targeting. Our results suggest a role for membrane lipid composition in targeting specificity. To see if the presence of an AH is a more general mechanism for SuP targeting, we searched for their presence within SuPs of both yeast and humans. Five endogenous yeast SuPs contained AHs, and of these four were sufficient for ER localization. Moreover, the presence of an AH was conserved to human SuP orthologs. By altering helix features we determine the parameters that affect this new targeting motif. Hence our work demonstrates how specific properties of AHs encode affinity for the ER membrane. More globally, understanding how SuPs are targeted correctly takes us a step forward in determining the underlying mechanisms of cellular localization and secretory pathway functions. The authors declare that they have no conflict of interest.

Here we analyzed the recycling of the Vps55/Vps68 complex in the yeast endocytic pathway. Deletion of VPS55 and VPS68 caused the same moderate stabilization of the endocytic cargo protein Ste6. No additive effect was observed by the double deletion, reinforcing the notion that both proteins form a functional unit. This is further underlined by the finding that the two proteins are dependent on each other for proper cellular localization. A tyrosine-based recycling signal was identified in the cytosolic tail of Vps68. Curiously, it turned out that the recycling signal was context dependent with respect to the usage of recycling factors. In its natural context, recycling was dependent on the sorting nexin Mvp1/SNX8 and independent of retromer. But, when the signal was inserted into a well-studied retromer substrate, the CPY receptor Vps10 devoid of its own signals, it became dependent on retromer and Snx3. This finding suggests that the availability of the recycling signal could be subjected to regulation. Previously, we obtained evidence that Vps68 cooperates with ESCRT-III in intraluminal vesicle (ILV) formation at late endosomes. It is thus conceivable that recycling of Vps55/Vps68 only occurs when its function in ILV formation is finished. Our data also suggest that recycling of Vps55/Vps68 could be regulated by phosphorylation. In addition, we identified Dcr2, the yeast orthologue of human sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A), as a new factor involved in Mvp1 dependent recycling of Vps55/Vps68. Article SummarySome proteins in the endocytic pathway are recycled to the Golgi or to the plasma membrane and are thus spared from degradation in the lysosome or vacuole. Here a recycling signal was identified in the Vps55/Vps68 complex. The data suggest that the accessibility of the signal is regulated. Further, a new factor involved in the sorting nexin Mvp1/SNX8 dependent recycling of Vps55/Vps68 was identified. This factor is Dcr2, the yeast orthologue of human sphingo-myelin phosphodiesterase acid-like 3A (SMPDL3A).

The organelles of yeast demonstrate diverse morphological traits in response to different stress stimuli. However, there is a lack of systematic reports on the structural changes induced by stress stimuli in all membrane-based organelles. Here, we utilized a set of fluorescent protein-based organelle markers to highlight the distinct characteristics of yeast under various stress triggers, including high temperature, hydrogen peroxide, acetic acid, and ethyl alcohol. We found that all of these organelles undergo alterations in structure or function in response to the four stress triggers we tested. Specifically, filamentous mitochondria rupture into smaller segments when exposed to the above four stress conditions. The structure of the endoplasmic reticulum (ER) remains relatively unchanged, but its function is affected. Additionally, high temperature and hydrogen peroxide can induce the Ire1p-mediated ER unfolded protein response (UPR). The translocation of most nuclear-localized proteins to the cytosol is dependent on the specific stress conditions employed. Under the above stress conditions, the vacuole undergoes fusion, resulting in the formation of a larger vacuole from multiple smaller ones. Meanwhile, acetic acid-induced stress leads to the translocation of vacuole-localized proteins Prc1p and Pep4p to unknown puncta, while Ybh3p relocates from the inner vacuole to the vacuole membrane. Proteins localized in the early Golgi, late Golgi, and late endosomes exhibit distinct traits, such as fading away or mis-localization. The structure and function of peroxisomes, lipid droplets, and autophagosomes also undergo modifications. Furthermore, upon exposure to high temperature and ethanol, apoptosis-related proteins Yca1, Aif1, and Mmi1 aggregate instead of remaining dispersed.

Many plasmids harbor unnecessary elements that complicate or hinder cloning tasks such as inserting one gene into another for protein domain grafting. In particular, restriction sites may be present in the backbone outside the polylinker region (multiple cloning site; MCS) and thus unavailable for use, and the overall length of a plasmid correlates with poorer ligation efficiency. To address these concerns, there has been a growing interest in minimal plasmids. Here, we describe the design and validation of a collection of six minimal integrating shuttle vectors for genetic manipulation in Saccharomyces cerevisiae. We constructed the plasmids using de novo gene synthesis and consisting only of a yeast selection marker (HIS3, TRP1, LEU2, URA3, natMX6, or KanMX), a bacterial selection marker (Ampicillin resistance), an origin of replication (ORI), and the MCS flanked by M13 forward and reverse sequences. We use truncated variants of these elements where available and eliminated all other sequences typically found in plasmids. The MCS consists of ten unique restriction sites. To our knowledge, at sizes ranging from approximately 2.6 kb to 3.5 kb, these are the smallest shuttle vectors described for yeast. Further, we removed common restriction sites in the open reading frames (ORFs) and terminators, freeing up approximately 30 cut sites in each plasmid. We named our pLS series in accordance with the well-known pRS vectors, which are on average 63% larger: pLS403 (HIS3), pLS404 (TRP1), pLS405 (LEU2), pLS406 (URA3), pLS408 (natMX6), and pLS410 (KanMX). These minimal vector backbones open up new opportunities for efficient molecular biology and genetic manipulation in Saccharomyces cerevisiae.

RILP is one of many effectors of RAB7 which bind to RAB7 in its activated GTP-bound state. The exact mechanism by which RAB7 effectors interact with RAB7 in time and space is not well understood. One of the known functions of RILP is to recruit dynein to RAB7-positive late endosomes. Dynein has been shown to be responsible for retrograde transport of RAB7-positive late endosomes in neuronal dendrites. We thus became interested in studying RILP in cultured neurons. We herein validate two commonly used anti-RILP antibodies which are commercially available. We find that both recognize only human RILP, but not mouse or rat RILP. These antibodies are thus not suitable for experiments carried out in mouse or rat cells. Furthermore, we find an unexpected difference between neurons and HEK293 cells in their ability to recruit overexpressed RILP to endosomes and cluster them in the cell center.

Although some budding yeasts have proved tractable and intensely studied models, others are more recalcitrant. Debaryomyces hansenii, an important yeast species in food and biotechnological industries with curious physiological characteristics, has proved difficult to manipulate genetically and remains poorly defined. To remedy this, we have combined live cell fluorescent dyes with high resolution imaging techniques to define the sub-cellular features of D. hansenii, such as the mitochondria, nuclei, vacuoles and the cell wall. Using these tools, we define biological processes like the cell cycle, organelle inheritance and different membrane trafficking pathways of D. hansenii for the first time. Beyond this, reagents designed to study Saccharomyces cerevisiae proteins were used to access proteomic information about D. hansenii. Finally, we optimised the use of label free holotomography to image yeast, defining the physical parameters and visualising sub-cellular features like membranes and vacuoles. Not only does this work shed light on D. hansenii but this combinatorial approach serves as a template for how other cell biological systems, which are not amenable to standard genetic procedures, can be studied.

Soluble secretory proteins with a signal peptide reach the extracellular space through the endoplasmic reticulum-Golgi conventional pathway. During translation, the signal peptide is recognised by the secretory recognition particle and results in a co-translational translocation to the endoplasmic reticulum to continue the secretory pathway. However, soluble secretory proteins lacking a signal peptide are also abundant, and several unconventional (endoplasmic reticulum/Golgi independent) pathways have been proposed and some demonstrated. This work describes new features of the secretion signal called N{beta}, originally identified in NaTrxh, a plant extracellular thioredoxin, that does not possess an orthodox signal peptide. We provide evidence that other proteins, including thioredoxins type h, with similar sequences are also signal peptide-lacking secretory proteins. To be a secretion signal, positions 5, 8 and 9 must contain neutral residues -a negative residue in position 9 in animal proteins- to maintain the N{beta} motif negatively charged and a hydrophilic profile. Moreover, our results suggest that the NaTrxh translocation to the endoplasmic reticulum occurs as a post-translational event. Finally, the N{beta} motif sequence at the N- or C-terminus could be a feature that may help to predict protein localisation, mainly in plant and animal proteins.

Exomer is a trans-Golgi protein complex involved in multiple biological processes, including lipid homeostasis and stress response. We have found that in fission yeast, the absence of exomer leads to a defect in the recovery of "target of rapamycin complex 2" (TORC2) activity in response to high concentrations of KCl, but not to sorbitol, which indicates that the mutants are impaired in their ability to respond to salt stress. Changes in lipid homeostasis did not suppress this defect. Ryh1RAB6 is a Rab GTPase that resides in the Golgi and endosomes and facilitates or stabilizes the interaction between TORC2 and its substrate, the AGC kinase Gad8. We have found genetic and functional interactions between exomer and Ryh1RAB6. The recovery of TORC2 activity in response to KCl requires Ryh1RAB6 and exomer, and the localization of Ryh1RAB6 is aberrant in exomer mutants. Co-immunoprecipitation experiments indicate that the interaction between TORC2 and Gad8 is weaker in the absence of exomer. We propose that exomer regulates TORC2 by facilitating appropriate Ryh1 localization.

The small GTPase Rho5 acts as a central hub to mediate the yeasts response to adverse environmental conditions, including oxidative stress, with the concomitant induction of mitophagy and apoptosis. A proper cellular stress response has been correlated with the rapid translocation of the GTPase to the mitochondria, which depends on its activating dimeric GDP/GTP exchange factor (GEF). Here, the small ALFA tag was attached to Rho5 or the GEF subunits Dck1 and Lmo1 to efficiently trap the functional fusion proteins to specific cellular membranes, i.e. the plasma membrane, the mitochon-drial outer membrane, or the nuclear membrane, via fusions of integral membrane proteins residing in these compartments with an ALFA nanobody. The trapped components were subjected to life-cell fluorescence microscopy in combination with GFP fusions of the GTPase or its GEF subunits to investigate their interaction in vivo. We found that the dimeric GEF tends to auto-assemble and form stable dimers independent of its intracellular localization. On the other hand, GFP-Rho5 does not stably colocalize with the trapped GEF, attributed to its transient interaction. Phenotypic analyses of strains with the misslocalized proteins indicate that for a proper oxidative stress response Lmo1 needs to associate with the plasma membrane. In contrast, Rho5 only exerts its role at the mitochondrial surface when it is there in its active conformation. These data underline the importance of the proper spatio-temporal distribution of Rho5-GTP during oxidative stress response.

Purine auxotrophy is a typical marker for many laboratory yeast strains. Supplementation of additional purine source (like adenine) is necessary to cultivate these strains. If not supplied in adequate amounts, purine starvation sets in. We tested purine starvation effects in budding yeast Saccharomyces cerevisiae ade8 knockout. We explored effects brought by purine starvation in cellular, central carbon metabolism and in the global transcriptome level. We observed that cells cultivated in purine depleted media became significantly more tolerant to severe thermal, oxidative and desiccation stresses when compared to the cells cultivated in media with all necessary supplements. When starved for purine, cells stop their cell cycle in G1 or G0 state; intracellular concentration of ATP, ADP and AMP decreases, but adenylate charge remains stable. Intracellular RNA concentration decreases and massive downregulation of ribosomal RNA occurs. We think that purine auxotrophic starvation in a way mimics "natural" nitrogen or carbon starvations and therefore initiates elements of a transcriptional program typical for stationary phase cells (cell cycle arrest, increased stress resistance). Therefore our results demonstrate that organised metabolic response is initiated not only via "natural starvations", but also when starving for metabolic intermediates, like purines.

Mitochondria contain their own genome (mtDNA), which is present in hundreds of copies per cell and organized into nucleoid structures that are distributed throughout the dynamic mitochondrial network. Beyond encoding essential protein subunits for oxidative phosphorylation, mtDNA can also serve as a signalling molecule when it is present into the cytosol. Despite the importance of this genome, there are still many unknowns with respect to its regulation. To study mtDNA dynamics in living cells, we have developed a genetic fluorescent reporter, mt-HI-NESS, which is based on the HI-NESS reporter that uses the bacterial H-NS DNA binding domain. Here, we describe how this reporter can be used to image mtDNA nucleoids for live cell imaging without affecting the replication or expression of the mtDNA. In addition to demonstrating the adaptability of the mt-HI-NESS reporter for multiple fluorescent proteins, we also emphasize important factors to consider during the optimization and application of this reporter.

Peroxisomes are ubiquitous cell organelles involved in various metabolic pathways. In order to properly function, several cofactors, substrates and products of peroxisomal enzymes need to pass the organellar membrane. So far only a few transporter proteins have been identified. We analysed peroxisomal membrane fractions purified from the yeast Hansenula polymorpha by untargeted label-free quantitation mass spectrometry. As expected, several known peroxisome-associated proteins were enriched in the peroxisomal membrane fraction. In addition, several other proteins were enriched, including mitochondrial transport proteins. Localization studies revealed that two of them, the mitochondrial carrier family proteins Aac2 and Mir1, have a dual localization on mitochondria and peroxisomes. To better understand the molecular mechanisms of dual sorting, we tested the localization of Mir1 in cells lacking Pex3 or Pex19, two peroxins that play a role in targeting of peroxisomal membrane proteins. In these cells Mir1 only localized to mitochondria, indicating that Pex3 and Pex19 are required to sort Mir1 to peroxisomes. Analysis of the localization of various truncated versions of Mir1 in wild-type H. polymorpha cells revealed that several localized to mitochondria, but only one, consisting of the transmembrane domains 3-6, was peroxisomal. Peroxisomal localization of this construct was lost in a MIR1 deletion strain, indicating that full length Mir1 was required for the localization of the truncated protein to peroxisomes. Our data suggest that only full length Mir1 sorts to peroxisomes, while Mir1 contains multiple regions with mitochondrial sorting information.

Bioluminescence resonance energy transfer (BRET) allows to quantitate protein interactions in intact cells. Here we provide a step-by-step protocol for measuring BRET due to transient interactions of oncogenic K-RasG12V in plasma membrane nanoclusters of HEK293-EBNA cells. We describe how to seed, transfect and replate cells, followed by their preparation for BRET-measurements on a microplate reader and detailed data analysis steps. For details on how to apply this protocol, please refer to Steffen et al., 2024 1. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/602189v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@152e8c2org.highwire.dtl.DTLVardef@2f2aacorg.highwire.dtl.DTLVardef@9a8e9borg.highwire.dtl.DTLVardef@108833f_HPS_FORMAT_FIGEXP M_FIG C_FIG

SYNOPSISThe AP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. Here, a synthetic reporter is presented that allows convenient monitoring of AP-3 traffic, and direct screening or selection for mutants with defects in the pathway. The reporter can be assayed by fluorescence microscopy or in liquid or agar plate formats and is adaptable to high-throughput screening. SUMMARYAP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. In mammals, mutations in AP-3 cause Hermansky-Pudlak Syndrome type 2, cyclic neutropenias, and a form of epileptic encephalopathy. In budding yeast, AP-3 carries cargo directly from the trans-Golgi to the lysosomal vacuole. Despite the pathways importance and its discovery two decades ago, rapid screens and selections for AP-3 mutants have not been available. We now report GNSI, a synthetic, genetically encoded reporter that allows rapid plate-based assessment of AP-3 functional deficiency, using either chromogenic or growth phenotype readouts. This system identifies defects in both the formation and consumption of AP-3 carrier vesicles and is adaptable to high-throughput screening or selection in both plate array and liquid batch culture formats. Episomal and integrating plasmids encoding GNSI have been submitted to the Addgene repository.

The Twin-arginine translocation (Tat) machinery, which is found in most cellular membranes containing a respiratory or photosynthetic electron transport chain, is characterized by its unique ability to catalyze membrane transport of folded proteins without impairing the membrane potential. In plant thylakoids, Tat machinery consists of three subunits, TatA, TatB, and TatC, with the latter two, TatB and TatC, forming membrane-integral multimeric TatBC receptor complexes. Here we have analyzed the stability and the subunit composition of these complexes after solubilization of thylakoids with the mild detergent digitonin as well as after additional affinity-purification. Employing different detergent combinations and/or heat treatment (40{degrees}C) followed by BN-PAGE and Western analysis we could identify four distinct Tat complexes with apparent molecular masses ranging from approximately 230 kDa to 620 kDa. Treatment of the largest Tat complex with either heat or detergents like DDM or Triton X-114 led to its stepwise breakdown into the three smaller complexes resulting from the successive release of TatB subunits from a relatively stable TatC core complex. From these data we conclude that the fully assembled, physiologically active TatBC receptor complex consists of a stable, trimeric TatC core to which three TatB subunits are bound independently from each other.