The Sec1 Family: A Novel Family of Proteins Involved in Synaptic Transmission and General Secretion

Abstract: The Sec1 family, a novel family of proteins involved in synaptic transmission and general secretion, is described. To date, 14 members of this family have been identified: four yeast proteins, Sec1, Sly1, Slp1/Vps33, and Vps45/Stt10; three nematode proteins, Unc-18 and the homologues of Sly1 and Slp1; the Drosophila Rop; and six mammalian proteins, the rat Munc-18/n-Sec1/rbSec1A and rbSec1B, the mouse Munc-18b/muSec1 and Munc-18c, and the bovine Munc-18 and mSec1. The mammalian proteins share 44–63% sequence identity with the nematode Unc-18 and Drosophila Rop proteins and 20–29% with the yeast proteins and their nematode homologues. The Sec1 proteins are mostly hydrophilic and lack a transmembrane domain. Nevertheless, Sec1 proteins are found as membrane-bound proteins. Some of them are also found as soluble, cytoplasmic proteins. Binding of the rat brain Sec1 to the presynaptic membrane may be due to strong interaction with syntaxin, an integral component of this membrane. The rat brain Sec1 is also bound to Cdk5, a neural cyclin-dependent kinase. The Sec1 proteins play a positive role in exocytosis. Loss of function mutations in SEC1 , SLY1 , or SLP1 result in blocking of protein transport between distinct yeast subcellular compartments. Inactivation of unc-18 and rop results in inhibition of neurotransmitter release and, in the case of rop , inhibition of general secretion as well. In addition, studies of Rop and n-Sec1 indicate that they also play a negative role in synaptic transmission, mediated by their interaction with syntaxin. A working model addressing the dual regulative role of the Sec1 proteins in secretion is presented.     

All roads lead to Rome (but some may be harder to travel): SRP-independent translocation into the endoplasmic reticulum

Abstract

Translocation into the endoplasmic reticulum (ER) is the first biogenesis step for hundreds of eukaryotic secretome proteins. Over the past 30 years, groundbreaking biochemical, structural and genetic studies have delineated one conserved pathway that enables ER translocation- the signal recognition particle (SRP) pathway. However, it is clear that this is not the only pathway which can mediate ER targeting and insertion. In fact, over the past decade, several SRP-independent pathways have been uncovered, which recognize proteins that cannot engage the SRP and ensure their subsequent translocation into the ER. These SRP-independent pathways face the same challenges that the SRP pathway overcomes: chaperoning the preinserted protein while in the cytosol, targeting it rapidly to the ER surface and generating vectorial movement that inserts the protein into the ER. This review strives to summarize the various mechanisms and machineries which mediate these stages of SRP-independent translocation, as well as examine why SRP-independent translocation is utilized by the cell. This emerging understanding of the various pathways utilized by secretory proteins to insert into the ER draws light to the complexity of the translocational task, and underlines that insertion into the ER might be more varied and tailored than previously appreciated.     

NDR2‐mediated Rabin8 phosphorylation is crucial for ciliogenesis by switching binding specificity from phosphatidylserine to Sec15

Primary cilia are antenna‐like sensory organelles protruding from the plasma membrane. Defects in ciliogenesis cause diverse genetic disorders. NDR2 was identified as the causal gene for a canine ciliopathy, early retinal degeneration, but its role in ciliogenesis remains unknown. Ciliary membranes are generated by transport and fusion of Golgi‐derived vesicles to the pericentrosome, a process requiring Rab11‐mediated recruitment of Rabin8, a GDP–GTP exchange factor (GEF) for Rab8, and subsequent Rab8 activation and Rabin8 binding to Sec15, a component of the exocyst that mediates vesicle tethering. This study shows that NDR2 phosphorylates Rabin8 at Ser‐272 and defects in this phosphorylation impair preciliary membrane assembly and ciliogenesis, resulting in accumulation of Rabin8‐/Rab11‐containing vesicles at the pericentrosome. Rabin8 binds to and colocalizes with GTP‐bound Rab11 and phosphatidylserine (PS) on pericentrosomal vesicles. The phospho‐mimetic S272E mutation of Rabin8 decreases affinity for PS but increases affinity for Sec15. These results suggest that NDR2‐mediated Rabin8 phosphorylation is crucial for ciliogenesis by triggering the switch in binding specificity of Rabin8 from PS to Sec15, thereby promoting local activation of Rab8 and ciliary membrane formation.     

A diterpenoid derivate compound targets selenocysteine of thioredoxin reductases and induces Bax/Bak-independent apoptosis

We have previously shown that the natural diterpenoid derivative S3 induced Bim upregulation and apoptosis in a Bax/Bak-independent manner. However, the exact molecular target(s) of S3 and the mechanism controlling Bim upregulation are still not clear. Here, we identify that S3 targets the selenoproteins TrxR1 and TrxR2 at the selenocysteine residue of the reactive center of the enzymes and inhibits their antioxidant activities. Consequently, cellular ROS is elevated, leading to the activation of FOXO3a, which contributes to Bim upregulation in Bax/Bak-deficient cells. Moreover, S3 retards tumor growth in subcutaneous xenograft tumors by inhibiting TrxR activity in vivo. Our studies delineate the signaling pathway controlling Bim upregulation, which results in Bax/Bak-independent apoptosis and provide evidence that the compounds can act as anticancer agents based on mammalian TrxRs inhibition.     

Sec62 Protein Mediates Membrane Insertion and Orientation of Moderately Hydrophobic Signal Anchor Proteins in the Endoplasmic Reticulum (ER)

Nascent chains are known to be targeted to the endoplasmic reticulum membrane either by a signal recognition particle (SRP)-dependent co-translational or by an SRP-independent post-translational translocation route depending on signal sequences. Using a set of model and cellular proteins carrying an N-terminal signal anchor sequence of controlled hydrophobicity and yeast mutant strains defective in SRP or Sec62 function, the hydrophobicity-dependent targeting efficiency and targeting pathway preference were systematically evaluated. Our results suggest that an SRP-dependent co-translational and an SRP-independent post-translational translocation are not mutually exclusive for signal anchor proteins and that moderately hydrophobic ones require both SRP and Sec62 for proper targeting and translocation to the endoplasmic reticulum. Further, defect in Sec62 selectively reduced signal sequences inserted in an N_in-C_out (type II) membrane topology, implying an undiscovered role of Sec62 in regulating the orientation of the signal sequence in an early stage of translocation.     

Three dimensional structure of the anthrax toxin translocon–lethal factor complex by cryo‐electron microscopy

We have visualized by cryo‐electron microscopy (cryo‐EM) the complex of the anthrax protective antigen (PA) translocon and the N‐terminal domain of anthrax lethal factor (LF_N) inserted into a nanodisc model lipid bilayer. We have determined the structure of this complex at a nominal resolution of 16 Å by single‐particle analysis and three‐dimensional reconstruction. Consistent with our previous analysis of negatively stained unliganded PA, the translocon comprises a globular structure (cap) separated from the nanodisc bilayer by a narrow stalk that terminates in a transmembrane channel (incompletely distinguished in this reconstruction). The globular cap is larger than the unliganded PA pore, probably due to distortions introduced in the previous negatively stained structures. The cap exhibits larger, more distinct radial protrusions, previously identified with PA domain three, fitted by elements of the NMFF PA prepore crystal structure. The presence of LF_N, though not distinguished due to the seven‐fold averaging used in the reconstruction, contributes to the distinct protrusions on the cap rim volume distal to the membrane. Furthermore, the lumen of the cap region is less resolved than the unliganded negatively stained PA, due to the low contrast obtained in our images of this specimen. Presence of the LF_N extended helix and N terminal unstructured regions may also contribute to this additional internal density within the interior of the cap. Initial NMFF fitting of the cryoEM‐defined PA pore cap region positions the Phe clamp region of the PA pore translocon directly above an internal vestibule, consistent with its role in toxin translocation.     

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein. We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n‐octyl‐oligo‐oxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,N‐dimethyldodecylamine N‐oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size‐dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore.     

Sec-dependent protein translocation across biological membranes: evolutionary conservation of an essential protein transport pathway (Review)

All living organisms, no matter how simple or complex, possess the ability to translocate proteins across biological membranes and into different cellular compartments. Although a range of membrane transport processes exist, the major pathway used to translocate proteins across the bacterial cytoplasmic membrane or the eukaryotic endoplasmic reticulum membrane is conserved and is known as the Sec or Sec61 pathway, respectively. Over the past two decades the Sec and Sec61 pathways have been studied extensively and are well characterised at the genetic and biochemical levels. However, it is only now with the recent structural determination of a number of the key elements of the pathways that the translocation complex is beginning to give up its secrets in exquisite molecular detail. This article will focus on the routes of Sec- and Sec61-dependent membrane targeting and the nature of the translocation channel in bacteria and eukaryotes.     

The type III secretion system needle,tip, and translocon

Many Gram‐negative bacteria pathogenic to plants and animals deploy the type III secretion system (T3SS) to inject virulence factors into their hosts. All bacteria that rely on the T3SS to cause infectious diseases in humans have developed antibiotic resistance. The T3SS is an attractive target for developing new antibiotics because it is essential in virulence, and part of its structural component is exposed on the bacterial surface. The structural component of the T3SS is the needle apparatus, which is assembled from over 20 different proteins and consists of a base, an extracellular needle, a tip, and a translocon. This review summarizes the current knowledge on the structure and assembly of the needle, tip, and translocon.