The Cas10 nuclease activity relieves host dormancy to facilitate spacer acquisition and retention during type III-A CRISPR immunity.

A hallmark of CRISPR immunity is the acquisition of short viral DNA sequences, known as spacers, that are transcribed into guide RNAs to recognize complementary sequences. The staphylococcal type III-A CRISPR-Cas system uses guide RNAs to locate viral transcripts and start a response that displays two mechanisms of immunity. When immunity is triggered by an early-expressed phage RNA, degradation of viral ssDNA can cure the host from infection. In contrast, when the RNA guide targets a late-expressed transcript, defense requires the activity of Csm6, a non-specific RNase. Here we show that Csm6 triggers a growth arrest of the host that provides immunity at the population level which hinders viral propagation to allow the replication of non-infected cells. We demonstrate that this mechanism leads to defense against not only the target phage but also other viruses present in the population that fail to replicate in the arrested cells. On the other hand, dormancy limits the acquisition and retention of spacers that trigger it. We found that the ssDNase activity of type III-A systems is required for the re-growth of a subset of the arrested cells, presumably through the degradation of the phage DNA, ending target transcription and inactivating the immune response. Altogether, our work reveals a built-in mechanism within type III-A CRISPR-Cas systems that allows the exit from dormancy needed for the subsistence of spacers that provide broad-spectrum immunity.

A systematic proximity ligation approach to studying protein-substrate specificity identifies the substrate spectrum of the Ssh1 translocon.

Many cellular functions are carried out by protein pairs or families, providing robustness alongside functional diversity. For such processes, it remains a challenge to map the degree of specificity versus promiscuity. Protein-protein interactions (PPIs) can be used to inform on these matters as they highlight cellular locals, regulation and, in cases where proteins affect other proteins - substrate range. However, methods to systematically study transient PPIs are underutilized. In this study, we create a novel approach to systematically compare stable or transient PPIs between two yeast proteins. Our approach, Cel-lctiv (CELlular biotin-Ligation for Capturing Transient Interactions in vivo), uses high-throughput pairwise proximity biotin ligation for comparing PPIs systematically and in vivo. As a proof of concept, we studied the homologous translocation pores Sec61 and Ssh1. We show how Cel-lctiv can uncover the unique substrate range for each translocon allowing us to pinpoint a specificity determinator driving interaction preference. More generally, this demonstrates how Cel-lctiv can provide direct information on substrate specificity even for highly homologous proteins.

Peroxisome function relies on organelle-associated mRNA translation.

Crucial metabolic functions of peroxisomes rely on a variety of peroxisomal membrane proteins (PMPs). While mRNA transcripts of PMPs were shown to be colocalized with peroxisomes, the process by which PMPs efficiently couple translation with targeting to the peroxisomal membrane remained elusive. Here, we combine quantitative electron microscopy with proximity-specific ribosome profiling and reveal that translation of specific PMPs occurs on the surface of peroxisomes in the yeast Saccharomyces cerevisiae. This places peroxisomes alongside chloroplasts, mitochondria, and the endoplasmic reticulum as organelles that use localized translation for ensuring correct insertion of hydrophobic proteins into their membranes. Moreover, the correct targeting of these transcripts to peroxisomes is crucial for peroxisomal and cellular function, emphasizing the importance of localized translation for cellular physiology.

Different modes of spacer acquisition by the Staphylococcus epidermidis type III-A CRISPR-Cas system.

CRISPR-Cas systems provide prokaryotic organisms with an adaptive defense mechanism that acquires immunological memories of infections. This is accomplished by integration of short fragments from the genome of invaders such as phages and plasmids, called 'spacers', into the CRISPR locus of the host. Depending on their genetic composition, CRISPR-Cas systems can be classified into six types, I-VI, however spacer acquisition has been extensively studied only in type I and II systems. Here, we used an inducible spacer acquisition assay to study this process in the type III-A CRISPR-Cas system of Staphylococcus epidermidis, in the absence of phage selection. Similarly to type I and II spacer acquisition, this type III system uses Cas1 and Cas2 to preferentially integrate spacers from the chromosomal terminus and free dsDNA ends produced after DNA breaks, in a manner that is enhanced by the AddAB DNA repair complex. Surprisingly, a different mode of spacer acquisition from rRNA and tRNA loci, which spans only the transcribed sequences of these genes and is not enhanced by AddAB, was also detected. Therefore, our findings reveal both common mechanistic principles that may be conserved in all CRISPR-Cas systems, as well as unique and intriguing features of type III spacer acquisition.

An ER surface retrieval pathway safeguards the import of mitochondrial membrane proteins in yeast.

The majority of organellar proteins are translated on cytosolic ribosomes and must be sorted correctly to function. Targeting routes have been identified for organelles such as peroxisomes and the endoplasmic reticulum (ER). However, little is known about the initial steps of targeting of mitochondrial proteins. In this study, we used a genome-wide screen in yeast and identified factors critical for the intracellular sorting of the mitochondrial inner membrane protein Oxa1. The screen uncovered an unexpected path, termed ER-SURF, for targeting of mitochondrial membrane proteins. This pathway retrieves mitochondrial proteins from the ER surface and reroutes them to mitochondria with the aid of the ER-localized chaperone Djp1. Hence, cells use the expanse of the ER surfaces as a fail-safe to maximize productive mitochondrial protein targeting.

Targeting and translocation of proteins to the endoplasmic reticulum at a glance.

The evolutionary emergence of organelles was a defining process in diversifying biochemical reactions within the cell and enabling multicellularity. However, compartmentalization also imposed a great challenge-the need to import proteins synthesized in the cytosol into their respective sites of function. For example, one-third of all genes encode for proteins that must be targeted and translocated into the endoplasmic reticulum (ER), which serves as the entry site to the majority of endomembrane compartments. Decades of research have set down the fundamental principles of how proteins get from the cytosol into the ER, and recent studies have brought forward new pathways and additional regulators enabling better definition of the rules governing substrate recognition. In this Cell Science at a Glance article and the accompanying poster, we give an overview of our current understanding of the multifaceted and regulated processes of protein targeting and translocation to the ER.

Multiple pathways facilitate the biogenesis of mammalian tail-anchored proteins.

Tail-anchored (TA) proteins are transmembrane proteins with a single C-terminal transmembrane domain, which functions as both their subcellular targeting signal and membrane anchor. We show that knockout of TRC40 in cultured human cells has a relatively minor effect on endogenous TA proteins, despite their apparent reliance on this pathway in vitro These findings support recent evidence that the canonical TRC40 pathway is not essential for TA protein biogenesis in vivo We therefore investigated the possibility that other ER-targeting routes can complement the TRC40 pathway and identified roles for both the SRP pathway and the recently described mammalian SND pathway in TA protein biogenesis. We conclude that, although TRC40 normally plays an important role in TA protein biogenesis, it is not essential, and speculate that alternative pathways for TA protein biogenesis, including those identified in this study, contribute to the redundancy of the TRC40 pathway.

hSnd2 protein represents an alternative targeting factor to the endoplasmic reticulum in human cells.

Recently, understanding of protein targeting to the endoplasmic reticulum (ER) was expanded by the discovery of multiple pathways that function in parallel to the signal recognition particle (SRP). Guided entry of tail-anchored proteins and SRP independent (SND) are two such targeting pathways described in yeast. So far, no human SND component is functionally characterized. Here, we report hSnd2 as the first constituent of the human SND pathway able to support substrate-specific protein targeting to the ER. Similar to its yeast counterpart, hSnd2 is assumed to function as a membrane-bound receptor preferentially targeting precursors carrying C-terminal transmembrane domains. Our genetic and physical interaction studies show that hSnd2 is part of a complex network of targeting and translocation that is dynamically regulated.

The SND proteins constitute an alternative targeting route to the endoplasmic reticulum.

In eukaryotes, up to one-third of cellular proteins are targeted to the endoplasmic reticulum, where they undergo folding, processing, sorting and trafficking to subsequent endomembrane compartments. Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40) and homologous yeast guided entry of tail-anchored proteins (GET) pathways. Despite the range of proteins that can be catered for by these two pathways, many proteins are still known to be independent of both SRP and GET, so there seems to be a critical need for an additional dedicated pathway for endoplasmic reticulum relay. We set out to uncover additional targeting proteins using unbiased high-content screening approaches. To this end, we performed a systematic visual screen using the yeast Saccharomyces cerevisiae, and uncovered three uncharacterized proteins whose loss affected targeting. We suggest that these proteins work together and demonstrate that they function in parallel with SRP and GET to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which we name Snd1, Snd2 and Snd3 (for SRP-independent targeting), can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system. This explains why it has previously been difficult to demonstrate complete loss of targeting for some substrates. Our discovery thus puts in place an essential piece of the endoplasmic reticulum targeting puzzle, highlighting how the targeting apparatus of the eukaryotic cell is robust, interlinked and flexible.

A cytosolic degradation pathway, prERAD, monitors pre-inserted secretory pathway proteins.

The endoplasmic reticulum (ER) identifies and disposes of misfolded secretory pathway proteins through the actions of ER-associated degradation (ERAD) pathways. It is becoming evident that a substantial fraction of the secretome transiently resides in the cytosol before translocating into the ER, both in yeast and in higher eukaryotes. To uncover factors that monitor this transient cytosolic protein pool, we carried out a genetic screen in Saccharomyces cerevisiae. Our findings highlighted a pre-insertional degradation mechanism at the cytosolic leaflet of the ER, which we term prERAD. prERAD relies on the concurrent action of the ER-localized ubiquitylation and deubiquitylation machineries Doa10 and Ubp1. By recognizing C-terminal hydrophobic motifs, prERAD tags for degradation pre-inserted proteins that have remained on the cytosolic leaflet of the ER for too long. Our discoveries delineate a new cellular safeguard, which ensures that every stage of secretory pathway protein biogenesis is scrutinized and regulated.

Embracing the void--how much do we really know about targeting and translocation to the endoplasmic reticulum?

In order for a protein to enter the secretory pathway, two crucial steps must occur: it first needs to be targeted to the cytosolic surface of the endoplasmic reticulum (ER), and then be translocated across the ER membrane. Although for many years studies of targeting focused on the signal recognition particle, recent findings reveal that several alternative targeting pathways exist, some still undescribed, and some only recently elucidated. In addition, many genes implicated in the translocation step have not been assigned a specific function. Here, we will focus on the open questions regarding ER targeting and translocation, and discuss how combining classical biochemistry with systematic approaches can promote our understanding of these essential cellular steps.