Knockout of SLy1 decreases double-negative thymocyte proliferation and protects mice from p53-induced tumor formation.

The lymphocyte-specific adapter protein SLy1 has previously been identified as indispensable for thymocyte development and T-cell proliferation and, recently, as a cause of X-linked combined immunodeficiency in humans that recapitulates many of the abnormalities reported in SLy1KO and SLy1d/d mice. As SLy1KO NK cells show increased levels of p53, we focused our research on the interdependency of SLy1 and p53 for thymocyte development. Using RT-PCR and immunoblot analysis, we observed increased levels of p53 as well as DNA damage response proteins in SLy1KO thymocytes. To test for rescue from SLy1-induced deficiencies in thymocyte development like reduced thymocyte numbers and reduced DN to DP progression, we generated a mouse model with T cell-specific p53-deficiency on an SLy1KO background and analyzed lymphocyte populations in these mice and respective controls. Astonishingly, SLy1KO -typical deficiencies were retained, showing that SLy1 is mechanistically independent of p53. Studies of apoptosis and proliferation in SLy1KO thymocytes revealed decreased proliferation in the DN3 subpopulation as a possible reason for the decreased thymocyte number. In mice with p53-deficient T cells, we observed tumor formation leading to reduced survival, preferentially in SLy1WT mice. Thus, we suggest that a SLy1-deficiency reduces proliferation, resulting in less hematologic tumors initiated by the p53-deficiency.

Arabidopsis retromer subunit AtVPS29 is involved in SLY1-mediated gibberellin signaling.

KEY MESSAGE: Retromer protein AtVPS29 upregulates the SLY1 protein and downregulates the RGA protein, positively stimulating the development of the root meristematic zone, which indicates an important role of AtVPS29 in gibberellin signaling. In plants, the large retromer complex is known to play roles in multiple development processes, including cell polarity, programmed cell death, and root hair growth in Arabidopsis. However, many of its roles in plant development remain unknown. Here, we show that Arabidopsis trimeric retromer protein AtVPS29 (vacuolar protein sorting 29) modulates gibberellin signaling. The SLEEPY1 (SLY1) protein, known as a positive regulator of gibberellic acid (GA) signaling, exhibited lower abundance in vps29-3 mutants compared to wild-type (WT) plants. Conversely, the DELLA repressor protein, targeted by the E3 ubiquitin ligase SCF (Skp, Cullin, F-box) complex and acting as a negative regulator of GA signaling, showed increased abundance in vps29-3 mutants compared to WT. The vps29-3 mutants exhibited decreased sensitivity to exogenous GA supply in contrast to WT, despite an upregulation in the expression of GA receptor genes within the vps29-3 mutants. In addition, the expression of the GA synthesis genes was downregulated in vps29-3 mutants, implying that the loss of AtVPS29 causes the downregulation of GA synthesis and signaling. Furthermore, vps29-3 mutants exhibited a reduced meristematic zone accompanied by a decreased cell number. Together, these data indicate that AtVPS29 positively regulates SLY1-mediated GA signaling and plant growth.

Multiple features within the syntaxin Sed5p mediate its Golgi localization.

Protein retention and the transport of proteins and lipids into and out of the Golgi is intimately linked to the biogenesis and homeostasis of this sorting hub of eukaryotic cells. Of particular importance are membrane proteins that mediate membrane fusion events with and within the Golgi-the Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). In the Golgi of budding yeast cells, the syntaxin SNARE Sed5p oversees membrane fusion events. Determining how Sed5p is localized to and trafficked within the Golgi is critical to informing our understanding of the mechanism(s) of biogenesis and homeostasis of this organelle. Here we establish that the steady-state localization of Sed5p to the Golgi appears to be primarily conformation-based relying on intra-molecular associations between the Habc domain and SNARE-motif while its tribasic COPI-coatomer binding motif plays a role in intra-Golgi retention.

Efficient ASK-assisted system for expression and purification of plant F-box proteins.

Ubiquitin-mediated protein degradation plays an essential role in plant growth and development as well as responses to environmental and endogenous signals. F-box protein is one of the key components of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which recruit specific substrate proteins for subsequent ubiquitination and 26S proteasome-mediated degradation to regulate developmental processes and signaling networks. However, it is not easy to obtain purified F-box proteins with high activity due to their unstable protein structures. Here, we found that Arabidopsis SKP-like proteins (ASKs) can significantly improve soluble expression of F-box proteins and maintain their bioactivity. We established an efficient ASK-assisted method to express and purify plant F-box proteins. The method meets a broad range of criteria required for the biochemical analysis or protein crystallization of plant F-box proteins.

SLy1 regulates T-cell proliferation during Listeria monocytogenes infection in a Foxo1-dependent manner.

Infection of mice with Listeria monocytogenes results in a strong T-cell response that is critical for an efficient defense. Here, we demonstrate that the adapter protein SLy1 (SH3-domain protein expressed in Lymphocytes 1) is essential for the generation of a fully functional T-cell response. The lack of SLy1 leads to reduced survival rates of infected mice. The increased susceptibility of SLy1 knock-out (KO) mice was caused by reduced proliferation of differentiated T cells. Ex vivo analyses of isolated SLy1 KO T cells displayed a dysregulation of Forkhead box protein O1 shuttling after TCR signaling, which resulted in an increased expression of cell cycle inhibiting genes, and therefore, reduced expansion of the T-cell population. Forkhead box protein O1 shuttles to the cytoplasm after phosphorylation in a protein complex including 14-3-3 proteins. Interestingly, we observed a similar regulation for the adapter protein SLy1, where TCR stimulation results in SLy1 phosphorylation and SLy1 export to the cytoplasm. Moreover, immunoprecipitation analyses revealed a binding of SLy1 to 14-3-3 proteins. Altogether, this study describes SLy1 as an immunoregulatory protein, which is involved in the generation of adaptive immune responses during L. monocytogenes infection, and provides a model of how SLy1 regulates T-cell proliferation.

E3 SUMO ligase AtSIZ1 positively regulates SLY1-mediated GA signalling and plant development.

Gibberellins affect various plant development processes including germination, cell division and elongation, and flowering. A large number of studies have been carried out to address the molecular mechanisms that mediate gibberellin signalling effects on plant growth. However, such studies have been limited to DELLA protein degradation; the regulatory mechanisms controlling how the stability and function of SLEEPY1 (SLY1), a protein that interacts with target DELLA proteins as components of the Skp, Cullin, F-box (SCF)(SLY1) complex, are modulated at the post-translational level have not been addressed. In the present study, we show that the E3 SUMO (small ubiquitin-related modifier) ligase AtSIZ1 regulates gibberellic acid signalling in Arabidopsis species by sumoylating SLY1. SLY1 was less abundant in siz1-2 mutants than in wild-type plants, but the DELLA protein repressor of ga1-3 (RGA) was more abundant in siz1-2 mutants than in wild-type plants. SLY1 also accumulated to a high level in the SUMO protease mutant esd4. Transgenic sly1-13 mutants over-expressing SLY1 were phenotypically similar to wild-type plants; however, sly1-13 plants over-expressing a mutated mSLY1 protein (K122R, a mutation at the sumoylation site) retained the mutant dwarfing phenotype. Over-expression of SLY1 in sly1-13 mutants resulted in a return of RGA levels to wild-type levels, but RGA accumulated to high levels in mutants over-expressing mSLY1. RGA was clearly detected in Arabidopsis co-expressing AtSIZ1 and mSLY1, but not in plants co-expressing AtSIZ1 and SLY1. In addition, sumoylated SLY1 interacted with RGA and SLY1 sumoylation was significantly increased by GA. Taken together, our results indicate that, in Arabidopsis, AtSIZ1 positively controls GA signalling through SLY1 sumoylation.

Loss of amyotrophic lateral sclerosis risk factor SCFD1 causes motor dysfunction in Drosophila.

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease mostly resulting from a complex interplay between genetic, environmental and lifestyle factors. Common genetic variants in the Sec1 Family Domain Containing 1 (SCFD1) gene have been associated with increased ALS risk in the most extensive genome-wide association study (GWAS). SCFD1 was also identified as a top-most significant expression Quantitative Trait Locus (eQTL) for ALS. Whether loss of SCFD1 function directly contributes to motor system dysfunction remains unresolved. Here we show that moderate gene silencing of Slh, the Drosophila orthologue of SCFD1, is sufficient to cause climbing and flight defects in adult flies. A more severe knockdown induced a significant reduction in larval mobility and profound neuromuscular junction (NMJ) deficits prior to death before metamorphosis. RNA-seq revealed downregulation of genes encoding chaperones that mediate protein folding downstream of Slh ablation. Our findings support the notion that loss of SCFD1 function is a meaningful contributor to ALS and disease predisposition may result from erosion of the mechanisms protecting against misfolding and protein aggregation.

Seed dormancy loss from dry after-ripening is associated with increasing gibberellin hormone levels in Arabidopsis thaliana.

Introduction: The seeds of many plants are dormant and unable to germinate at maturity, but gain the ability to germinate through after-ripening during dry storage. The hormone abscisic acid (ABA) stimulates seed dormancy, whereas gibberellin A (GA) stimulates dormancy loss and germination. Methods: To determine whether dry after-ripening alters the potential to accumulate ABA and GA, hormone levels were measured during an after-ripening time course in dry and imbibing ungerminated seeds of wildtype Landsberg erecta (Ler) and of the highly dormant GA-insensitive mutant sleepy1-2 (sly1-2). Results: The elevated sly1-2 dormancy was associated with lower rather than higher ABA levels. Ler germination increased with 2-4 weeks of after-ripening whereas sly1-2 required 21 months to after-ripen. Increasing germination capacity with after-ripening was associated with increasing GA4 levels in imbibing sly1-2 and wild-type Ler seeds. During the same 12 hr imbibition period, after-ripening also resulted in increased ABA levels. Discussion: The decreased ABA levels with after-ripening in other studies occurred later in imbibition, just before germination. This suggests a model where GA acts first, stimulating germination before ABA levels decline, and ABA acts as the final checkpoint preventing germination until processes essential to survival, like DNA repair and activation of respiration, are completed. Overexpression of the GA receptor GID1b (GA INSENSITIVE DWARF1b) was associated with increased germination of sly1-2 but decreased germination of wildtype Ler. This reduction of Ler germination was not associated with increased ABA levels. Apparently, GID1b is a positive regulator of germination in one context, but a negative regulator in the other.

An imaging-based RNA interference screen for modulators of the Rab6-mediated Golgi-to-ER pathway in mammalian cells.

In mammalian cells, membrane traffic pathways play a critical role in connecting the various compartments of the endomembrane system. Each of these pathways is highly regulated, requiring specific machinery to ensure their fidelity. In the early secretory pathway, transport between the endoplasmic reticulum (ER) and Golgi apparatus is largely regulated via cytoplasmic coat protein complexes that play a role in identifying cargo and forming the transport carriers. The secretory pathway is counterbalanced by the retrograde pathway, which is essential for the recycling of molecules from the Golgi back to the ER. It is believed that there are at least two mechanisms to achieve this - one using the cytoplasmic COPI coat complex, and another, poorly characterised pathway, regulated by the small GTPase Rab6. In this work, we describe a systematic RNA interference screen targeting proteins associated with membrane fusion, in order to identify the machinery responsible for the fusion of Golgi-derived Rab6 carriers at the ER. We not only assess the delivery of Rab6 to the ER, but also one of its cargo molecules, the Shiga-like toxin B-chain. These screens reveal that three proteins, VAMP4, STX5, and SCFD1/SLY1, are all important for the fusion of Rab6 carriers at the ER. Live cell imaging experiments also show that the depletion of SCFD1/SLY1 prevents the membrane fusion event, suggesting that this molecule is an essential regulator of this pathway.

Isolation and Role of PmRGL2 in GA-mediated Floral Bud Dormancy Release in Japanese Apricot (Prunus mume Siebold et Zucc.).

Bud dormancy release is regulated by gibberellins (GAs). DELLA proteins are highly conserved and act as negative regulators in GA signaling pathway. The present study established a relationship between PmRGL2 in Japanese apricot and GA4 levels during dormancy release of floral buds. Overexpression of PmRGL2 in poplar delayed the onset of bud dormancy and resulted in dwarf plants, relative to wild-type trees. PmRGL2 exhibited higher expression during ecodormancy and relatively lower expression during endodormancy. The relative level of GA4 exhibited an increasing trend at the transition from endodormancy to ecodormancy and displayed a similar expression pattern of genes related to GA metabolism, PmGA20ox2, PmGA3ox1, PmGID1b, in both Japanese apricot and transgenic poplar. These results suggests that PmRGL2 acts as an integrator and negative regulator of dormancy via a GA-signaling pathway. Moreover, an interaction between RGL2 and SLY1 in a yeast two hybrid (Y2H) system further suggests that SCF E3 ubiquitin ligases, such as SLY1, may be a critical factor in the regulation of RGL2 through an SCF SLY1 -proteasome pathway. Our study demonstrated that PmRGL2 plays a negative role in bud dormancy release by regulating the GA biosynthetic enzymes, GA20ox and GA3ox1 and the GA receptor, GID1b.

Biology in the Dry Seed: Transcriptome Changes Associated with Dry Seed Dormancy and Dormancy Loss in the Arabidopsis GA-Insensitive sleepy1-2 Mutant.

Plant embryos can survive years in a desiccated, quiescent state within seeds. In many species, seeds are dormant and unable to germinate at maturity. They acquire the capacity to germinate through a period of dry storage called after-ripening (AR), a biological process that occurs at 5-15% moisture when most metabolic processes cease. Because stored transcripts are among the first proteins translated upon water uptake, they likely impact germination potential. Transcriptome changes associated with the increased seed dormancy of the GA-insensitive sly1-2 mutant, and with dormancy loss through long sly1-2 after-ripening (19 months) were characterized in dry seeds. The SLY1 gene was needed for proper down-regulation of translation-associated genes in mature dry seeds, and for AR up-regulation of these genes in germinating seeds. Thus, sly1-2 seed dormancy may result partly from failure to properly regulate protein translation, and partly from observed differences in transcription factor mRNA levels. Two positive regulators of seed dormancy, DELLA GAI (GA-INSENSITIVE) and the histone deacetylase HDA6/SIL1 (MODIFIERS OF SILENCING1) were strongly AR-down-regulated. These transcriptional changes appeared to be functionally relevant since loss of GAI function and application of a histone deacetylase inhibitor led to decreased sly1-2 seed dormancy. Thus, after-ripening may increase germination potential over time by reducing dormancy-promoting stored transcript levels. Differences in transcript accumulation with after-ripening correlated to differences in transcript stability, such that stable mRNAs appeared AR-up-regulated, and unstable transcripts AR-down-regulated. Thus, relative transcript levels may change with dry after-ripening partly as a consequence of differences in mRNA turnover.

Binding of GID1 to DELLAs promotes dissociation of GAF1 from DELLA in GA dependent manner.

Gibberellins (GAs) are important phytohormones for plant growth and development. DELLAs are members of the plant-specific GRAS protein family and act as repressors of GA signaling. DELLAs are rapidly degraded in the presence of GAs. GA-GID1-DELLA complexes are recognized and ubiquitinated by the SCF(SLY) complex. The sleepy1 (sly1) F-box mutant exhibits dwarfism and low-germination phenotypes due to high accumulation of DELLAs. Overexpression of GID1 in the sly1 mutant partially rescues these phenotypes without degradation of DELLAs suggesting that proteolysis independent regulation of DELLAs exists in GA signaling. But the molecular mechanisms of non-proteolytic regulation of DELLA are largely unknown. Recently we identified a DELLA binding transcription factor, GAI-ASSOCIATED FACTOR1 (GAF1). GAF1 also interacts with co-repressor TOPLESS RELATED (TPR) in nuclei. DELLAs and TPR act as coactivator and corepressor of GAF1, respectively. GAs converts the GAF1 complex from transcriptional activator to repressor via degradation of DELLAs. The overexpression of ΔPAM, lacking of DELLAs binding region of GAF1, partially rescue dwarf phenotypes of GA deficient or GA insensitive mutant. In this study, we investigate the relationship between non-proteolytic regulation of DELLAs and GA signaling via DELLA-GAF1 complex using modified yeast two-hybrid system.

The roles of the GA receptors GID1a, GID1b, and GID1c in sly1-independent GA signaling.

Gibberellin (GA) hormone signaling occurs through proteolytic and non-proteolytic mechanisms. GA binding to the GA receptor GID1 (GA-INSENSITIVE DWARF1) enables GID1 to bind negative regulators of GA responses called DELLA proteins. In proteolytic GA signaling, the SLEEPY1 (SLY1) F-box protein targets DELLA proteins in the GID1-GA-DELLA complex for destruction through the ubiquitin-proteasome pathway. Non-proteolytic GA signaling in sly1 mutants where GA cannot target DELLA proteins for destruction, requires GA and GID1 gene function. Based on comparison of gid1 multiple mutants to sly1 gid1 mutants, GID1a is the primary GA receptor stimulating stem elongation in proteolytic and non-proteolytic signaling, and stimulating fertility in proteolytic GA signaling. GID1b plays the primary role in fertility, and a secondary role in elongation during non-proteolytic GA signaling. The stronger role of GID1b in non-proteolytic GA signaling may result from the fact that GID1b has higher affinity for DELLA protein than GID1a and GID1c.

SLY1 and Syntaxin 18 specify a distinct pathway for procollagen VII export from the endoplasmic reticulum.

TANGO1 binds and exports Procollagen VII from the endoplasmic reticulum (ER). In this study, we report a connection between the cytoplasmic domain of TANGO1 and SLY1, a protein that is required for membrane fusion. Knockdown of SLY1 by siRNA arrested Procollagen VII in the ER without affecting the recruitment of COPII components, general protein secretion, and retrograde transport of the KDEL-containing protein BIP, and ERGIC53. SLY1 is known to interact with the ER-specific SNARE proteins Syntaxin 17 and 18, however only Syntaxin 18 was required for Procollagen VII export. Neither SLY1 nor Syntaxin 18 was required for the export of the equally bulky Procollagen I from the ER. Altogether, these findings reveal the sorting of bulky collagen family members by TANGO1 at the ER and highlight the existence of different export pathways for secretory cargoes one of which is mediated by the specific SNARE complex containing SLY1 and Syntaxin 18.DOI: http://dx.doi.org/10.7554/eLife.02784.001.