The spatiotemporal control of cell polarity is crucial for the development of multicellular organisms and for reliable polarity switches during cell cycle progression in unicellular systems. A tight control of cell polarity is especially important in haploid budding yeast, where the new polarity site (bud site) is established next to the cell division site after cell separation. How cells coordinate the temporal establishment of two adjacent polarity sites remains elusive. Here, we report that the bud neck associated protein Gps1 (GTPase-mediated polarity switch 1) establishes a novel polarity cue that concomitantly sustains Rho1-dependent polarization and inhibits premature Cdc42 activation at the site of cytokinesis. Failure of Gps1 regulation leads to daughter cell death due to rebudding inside the old bud site. Our findings provide unexpected insights into the temporal control of cytokinesis and describe the importance of a Gps1-dependent mechanism for highly accurate polarity switching between two closely connected locations.

Despite the critical contributions of cilia to embryonic development and human health, key regulators of cilia formation await identification. In this paper, a functional RNA interference-based screen linked 30 novel protein kinases with ciliogenesis. Of them, we have studied the role of the microtubule (MT)-associated protein/MT affinity regulating kinase 4 (MARK4) in depth. MARK4 associated with the basal body and ciliary axoneme in human and murine cell lines. Ultrastructural and functional analyses established that MARK4 kinase activity was required for initiation of axoneme extension. We identified the mother centriolar protein ODF2 as an interaction partner of MARK4 and showed that ODF2 localization to the centriole partially depended on MARK4. Our data indicated that, upon MARK4 or ODF2 knockdown, the ciliary program arrested before the complete removal of the CP110-Cep97 inhibitory complex from the mother centriole, suggesting that these proteins act at this level of axonemal extension. We propose that MARK4 is a critical positive regulator of early steps in ciliogenesis.

Gene tagging facilitates systematic genomic and proteomic analyses but chromosomal tagging typically disrupts gene regulatory sequences. Here we describe a seamless gene tagging approach that preserves endogenous gene regulation and is potentially applicable in any species with efficient DNA double-strand break repair by homologous recombination. We implement seamless tagging in Saccharomyces cerevisiae and demonstrate its application for protein tagging while preserving simultaneously upstream and downstream gene regulatory elements. Seamless tagging is compatible with high-throughput strain construction using synthetic genetic arrays (SGA), enables functional analysis of transcription antisense to open reading frames and should facilitate systematic and minimally-invasive analysis of gene functions.

Single ribonucleoside monophosphates (rNMPs) are transiently present in eukaryotic genomes. The RNase H2-dependent ribonucleotide excision repair (RER) pathway ensures error-free rNMP removal. In some pathological conditions, rNMP removal is impaired. If these rNMPs hydrolyze during, or prior to, S phase, toxic single-ended double-strand breaks (seDSBs) can occur upon an encounter with replication forks. How such rNMP-derived seDSB lesions are repaired is unclear. We expressed a cell cycle phase restricted allele of RNase H2 to nick at rNMPs in S phase and study their repair. Although Top1 is dispensable, the RAD52 epistasis group and Rtt101Mms1-Mms22 dependent ubiquitylation of histone H3 become essential for rNMP-derived lesion tolerance. Consistently, loss of Rtt101Mms1-Mms22 combined with RNase H2 dysfunction leads to compromised cellular fitness. We refer to this repair pathway as nick lesion repair (NLR). The NLR genetic network may have important implications in the context of human pathologies.

Selective protein degradation typically involves substrate recognition via short linear motifs known as degrons. Various degrons can be found at protein termini from bacteria to mammals. While N-degrons have been extensively studied, our understanding of C-degrons is still limited. Towards a comprehensive understanding of eukaryotic C-degron pathways, here we perform an unbiased survey of C-degrons in budding yeast. We identify over 5000 potential C-degrons by stability profiling of random peptide libraries and of the yeast C‑terminome. Combining machine learning, high-throughput mutagenesis and genetic screens reveals that the SCF ubiquitin ligase targets ~40% of degrons using a single F-box substrate receptor Das1. Although sequence-specific, Das1 is highly promiscuous, recognizing a variety of C-degron motifs. By screening for full-length substrates, we implicate SCFDas1 in degradation of orphan protein complex subunits. Altogether, this work highlights the variety of C-degron pathways in eukaryotes and uncovers how an SCF/C-degron pathway of broad specificity contributes to proteostasis.

Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs.

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

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.

Most eukaryotic proteins are N-terminally acetylated. This modification can be recognized as a signal for selective protein degradation (degron) by the N-end rule pathways. However, the prevalence and specificity of such degrons in the proteome are unclear. Here, by systematically examining how protein turnover is affected by N-terminal sequences, we perform a comprehensive survey of degrons in the yeast N-terminome. We find that approximately 26% of nascent protein N termini encode cryptic degrons. These degrons exhibit high hydrophobicity and are frequently recognized by the E3 ubiquitin ligase Doa10, suggesting a role in protein quality control. In contrast, N-terminal acetylation rarely functions as a degron. Surprisingly, we identify two pathways where N-terminal acetylation has the opposite function and blocks protein degradation through the E3 ubiquitin ligase Ubr1. Our analysis highlights the complexity of N-terminal degrons and argues that hydrophobicity, not N-terminal acetylation, is the predominant feature of N-terminal degrons in nascent proteins.

The chitin synthase Chs3 is a multipass membrane protein whose trafficking is tightly controlled. Accordingly, its exit from the endoplasmic reticulum (ER) depends on several complementary mechanisms that ensure its correct folding. Despite its potential failure on its exit, Chs3 is very stable in this compartment, which suggests its poor recognition by ER quality control mechanisms such as endoplasmic reticulum-associated degradation (ERAD). Here we show that proper N-glycosylation of its luminal domain is essential to prevent the aggregation of the protein and its subsequent recognition by the Hrd1-dependent ERAD-L machinery. In addition, the interaction of Chs3 with its chaperone Chs7 seems to mask additional cytosolic degrons, thereby avoiding their recognition by the ERAD-C pathway. On top of that, Chs3 molecules that are not degraded by conventional ERAD can move along the ER membrane to reach the inner nuclear membrane, where they are degraded by the inner nuclear membrane-associated degradation (INMAD) system, which contributes to the intracellular homeostasis of Chs3. These results indicate that Chs3 is an excellent model to study quality control mechanisms in the cell and reinforce its role as a paradigm in intracellular trafficking research.

Cdc42 is a highly conserved master regulator of cell polarity. Here, we investigated the mechanism by which yeast cells never re-establish polarity at cortical sites (cytokinesis remnants [CRMs]) that have previously supported Cdc42-mediated growth as a paradigm to mechanistically understand how Cdc42-inhibitory polarity cues are established. We revealed a two-step mechanism of loading the Cdc42 antagonist Nba1 into CRMs to mark these compartments as refractory for a second round of Cdc42 activation. Our data indicate that Nba1 together with a cortically tethered adaptor protein confers memory of previous polarization events to translate this spatial legacy into a biochemical signal that ensures the local singularity of Cdc42 activation. "Memory loss" mutants that repeatedly use the same polarity site over multiple generations display nuclear segregation defects and a shorter lifespan. Our work thus established CRMs as negative polarity cues that prevent Cdc42 reactivation to sustain the fitness of replicating cells.

The spatiotemporal control of mitotic exit is crucial for faithful chromosome segregation during mitosis. In budding yeast, the mitotic exit network (MEN) drives cells out of mitosis, whereas the spindle position checkpoint (SPOC) blocks MEN activity when the anaphase spindle is mispositioned. How the SPOC operates at a molecular level remains unclear. Here, we report novel insights into how mitotic signalling pathways orchestrate chromosome segregation in time and space. We establish that the key function of the central SPOC kinase, Kin4, is to counterbalance MEN activation by the cdc fourteen early anaphase release (FEAR) network in the mother cell compartment. Remarkably, Kin4 becomes dispensable for SPOC function in the absence of FEAR. Cells lacking both FEAR and Kin4 show that FEAR contributes to mitotic exit through regulation of the SPOC component Bfa1 and the MEN kinase Cdc15. Furthermore, we uncover controls that specifically promote mitotic exit in the daughter cell compartment.

The proteome of mitochondria comprises mostly proteins that originate as precursors in the cytosol. Before import into the organelle, such proteins are exposed to cytosolic quality control mechanisms. Multiple lines of evidence indicate a significant contribution of the major cytosolic protein degradation machinery, the ubiquitin-proteasome system, to the quality control of mitochondrial proteins. Proteins that are directed to the mitochondrial intermembrane space (IMS) exemplify an entire class of mitochondrial proteins regulated by proteasomal degradation. However, little is known about how these proteins are selected for degradation.

Cilia formation is a multi-step process that starts with the docking of a vesicle at the distal part of the mother centriole. This step marks the conversion of the mother centriole into the basal body, from which axonemal microtubules extend to form the ciliary compartment. How vesicles are stably attached to the mother centriole to initiate ciliary membrane biogenesis is unknown. Here, we investigate the molecular role of the mother centriolar component Cep164 in ciliogenesis. We show that Cep164 was indispensable for the docking of vesicles at the mother centriole. Using biochemical and functional assays, we identified the components of the vesicular transport machinery, the GEF Rabin8 and the GTPase Rab8, as interacting partners of Cep164. We propose that Cep164 is targeted to the apical domain of the mother centriole to provide the molecular link between the mother centriole and the membrane biogenesis machinery that initiates cilia formation.

Stable unannotated transcripts (SUTs), some of which overlap protein-coding genes in antisense direction, are a class of non-coding RNAs. While case studies have reported important regulatory roles for several of such RNAs, their general impact on protein abundance regulation of the overlapping gene is not known. To test this, we employed seamless gene manipulation to repress antisense SUTs of 162 yeast genes by using a unidirectional transcriptional terminator and a GFP tag. We found that the mere presence of antisense SUTs was not sufficient to influence protein abundance, that observed effects of antisense SUTs correlated with sense transcript start site overlap, and that the effects were generally weak and led to reduced protein levels. Antisense regulated genes showed increased H3K4 di- and trimethylation and had slightly lower than expected noise levels. Our results suggest that the functionality of antisense RNAs has gene and condition-specific components.

The yeast Saccharomyces cerevisiae is ideal for systematic studies relying on collections of modified strains (libraries). Despite the significance of yeast libraries and the immense variety of available tags and regulatory elements, only a few such libraries exist, as their construction is extremely expensive and laborious. To overcome these limitations, we developed a SWAp-Tag (SWAT) method that enables one parental library to be modified easily and efficiently to give rise to an endless variety of libraries of choice. To showcase the versatility of the SWAT approach, we constructed and investigated a library of ∼1,800 strains carrying SWAT-GFP modules at the amino termini of endomembrane proteins and then used it to create two new libraries (mCherry and seamless GFP). Our work demonstrates how the SWAT method allows fast and effortless creation of yeast libraries, opening the door to new ways of systematically studying cell biology.

The conserved NDR-kinase Dbf2 plays a critical role in cytokinesis in budding yeast. Among its cytokinesis-related substrates is the F-BAR protein Hof1. Hof1 colocalizes at the cell division site with the septin complex and, as mitotic exit progresses, moves to the actomyosin ring (AMR). Neither the function of Hof1 at the septin complex nor the mechanism by which Hof1 supports AMR constriction is understood. Here we establish that Dbf2 has a dual function in Hof1 regulation. First, we show that the coiled-coil region, which is adjacent to the conserved F-BAR domain, is required for the binding of Hof1 to septins. The Dbf2-dependent phosphorylation of Hof1 at a single serine residue (serine 313) in this region diminishes the recruitment of Hof1 to septins both in vitro and in vivo. Genetic and functional analysis indicates that the binding of Hof1 to septins is important for septin rearrangement and integrity during cytokinesis. Furthermore, Dbf2 phosphorylation of Hof1 at serines 533 and 563 promotes AMR constriction most likely by inhibiting the SH3-domain-dependent interactions of Hof1. Thus our data show that Dbf2 coordinates septin and AMR functions during cytokinesis through the regulation/control of Hof1.

N-terminal acetylation is a prominent protein modification, and inactivation of N-terminal acetyltransferases (NATs) cause protein homeostasis stress. Using multiplexed protein stability profiling with linear ubiquitin fusions as reporters for the activity of the ubiquitin proteasome system, we observed increased ubiquitin proteasome system activity in NatA, but not NatB or NatC mutants. We find several mechanisms contributing to this behavior. First, NatA-mediated acetylation of the N-terminal ubiquitin-independent degron regulates the abundance of Rpn4, the master regulator of the expression of proteasomal genes. Second, the abundance of several E3 ligases involved in degradation of UFD substrates is increased in cells lacking NatA. Finally, we identify the E3 ligase Tom1 as a novel chain-elongating enzyme (E4) involved in the degradation of linear ubiquitin fusions via the formation of branched K11, K29, and K48 ubiquitin chains, independently of the known E4 ligases involved in UFD, leading to enhanced ubiquitination of the UFD substrates.

Faithful chromosome segregation in budding yeast requires correct positioning of the mitotic spindle along the mother to daughter cell polarity axis. When the anaphase spindle is not correctly positioned, a surveillance mechanism, named as the spindle position checkpoint (SPOC), prevents the progression out of mitosis until correct spindle positioning is achieved. How SPOC works on a molecular level is not well understood. Here we performed a genome-wide genetic screen to search for components required for SPOC. We identified the SWR1 chromatin-remodeling complex (SWR1-C) among several novel factors that are essential for SPOC integrity. Cells lacking SWR1-C were able to activate SPOC upon spindle misorientation but underwent mitotic slippage upon prolonged SPOC arrest. This mitotic slippage required the Cdc14-early anaphase release pathway and other factors including the SAGA (Spt-Ada-Gcn5 acetyltransferase) histone acetyltransferase complex, proteasome components and the mitotic cyclin-dependent kinase inhibitor Sic1. Together, our data establish a novel link between SWR1-C chromatin remodeling and robust checkpoint arrest in late anaphase.