Stent Retriever Deployment Tracing Susceptibility Vessel Sign in the M2 Branch Predicts the Effective First-Pass Reperfusion in Thrombectomy for M1 Occlusion.

BACKGROUND AND PURPOSE: Successful first-pass reperfusion is associated with better functional outcomes after mechanical thrombectomy (MT) for acute ischemic stroke, but its treatment strategies remain unclear. MATERIALS AND METHODS: We retrospectively recruited patients who underwent MT for M1 occlusion between December 2020 and May 2023 at our institution. The locations of susceptibility vessel sign (SVS) on magnetic resonance imaging were classified into M1 only, M1 to single M2 branch, or M1 to both M2 branches. Patients were included in the SVS tracing group when the stent retriever of the first pass covered the entire SVS length. Successful reperfusion was defined as a modified Thrombolysis in Cerebral Infarction scale 2b-3. Any intracranial hemorrhage detected at 24-hour postoperatively was included as a hemorrhagic complication. RESULTS: The SVS was detected in M1 only, M1 to single M2 branch, and M1 to both M2 branches in 8, 22, and 4 patients, respectively. Among the 34 patients, 27 were included in the SVS-tracing group. Successful first-pass reperfusion was significantly more frequent in the SVS-tracing group compared with the non-SVS tracing group (odds ratio, 14.4; 95% confidence interval, 2.0 - 101; P = 0.007). The procedural time was significantly reduced in the SVS tracing group (median, 29 [interquartile range, 22 - 49] minute vs. 63 [43 - 106] minute; P = 0.043). There was a trend toward less frequent hemorrhagic complications in the SVS tracing group (odds ratio, 0.17; 95% confidence interval, 0.029 - 1.0; P = 0.052). CONCLUSIONS: This study provides a thrombus imaging-based MT strategy to efficiently achieve first-pass reperfusion in M1 occlusion.

Preoperative Ocular Symptoms Predict Acute Glaucoma After Carotid Revascularization: An Analysis of Combined Single-Center Data and a Systematic Review.

BACKGROUND: Acute glaucoma is a potential complication of carotid revascularization procedures such as endarterectomy or stenting. Although preoperative ocular hypoperfusion may predispose patients to postoperative glaucoma, the details of this complication have not been clarified. METHODS: We retrospectively reviewed the medical records of consecutive patients who underwent carotid revascularization at our institution from January 2019 to December 2022. These patients were divided into glaucoma and nonglaucoma groups. Given the rarity of the event, a systematic literature review was performed to additionally include data from patients who developed acute glaucoma after carotid revascularization. Multivariate logistic regression was performed to identify the risk factors for acute glaucoma. RESULTS: Thirty-five cases, including 2 from our institution, were included in the glaucoma group, and 130 were included in the nonglaucoma group. Most cases (79%) occurred within five days postoperatively. Multivariate analysis revealed that preoperative ocular symptoms were significantly associated with the development of postoperative glaucoma (odds ratio, 361.06; 95% confidence interval, 34.09-3824.27; P < 0.001). Preoperative neovascularization at the iris or anterior chamber angle, indicating severe ocular hypoperfusion, was found in 84% of patients with glaucoma. Permanent visual loss occurred in 41% of patients. The incidence of postoperative glaucoma at our institution was 1.5% (2/132). The positive predictive value of preoperative ocular symptoms for postoperative glaucoma was 0.25 (95% confidence interval, 0.18-0.32). CONCLUSIONS: This study was the first to clarify the risk factors and characteristics of acute glaucoma after carotid revascularization.

Japan Trevo Registry: Real-world Registry of Stent Retriever Alone or in Combined Therapy with Aspiration Catheter for Acute Ischemic Stroke in Japan.

Endovascular therapy (EVT) for real-world patients after extended time frames is associated with concerns about its efficacy and safety. We conducted a prospective registry at 77 centers between November 2019 and October 2020. The registry criteria included patients treated with Trevo Retriever alone or in combined therapy with an aspiration catheter. The primary outcome was effective reperfusion (thrombolysis in cerebral infarction grade ≥ 2b), the secondary outcome was a modified Rankin scale 0-2 at 90 days, and the safety outcomes were worsening of neurologic symptoms within 24 h postoperatively, intracranial hemorrhage (ICH) within 24 h after EVT and mortality. We also exlpored the difference between patients whose last known well time (LKWT) to a puncture was less than 6 h (0-6 h) and those whose LKWT was 6 h or more but less than 24 h (6-24 h). Among the 1041 patients registered, 1025 patients were analyzed. The mean age was 76.9 years, and 53.6% of the participants were males. The 6-24 h group was 206/998 (20.6%), the median National Institute of Health Stroke Scale (NIHSS) score at admission was 18, and the median Alberta Stroke Program Early CT score was 8. Combined technique as the first pass was used on 817 (79.7%) patients. The primary outcome was 934 (91.1%). The secondary outcome was 433/1021 (42.4%). Symptomatic ICH, any ICH, and mortality were 10/1019 (1.0%), 311/1019 (30.5%), and 75 (7.3%). In the subanalysis, the 6-24 h group was lower in NIHSS (median;18 vs 16), and the secondary outcome was not significantly different in the <6 h group. Even after treatment time expansion, this result was comparable to other Trevo-based trials and nationwide registries.

S2P intramembrane protease RseP degrades small membrane proteins and suppresses the cytotoxicity of intrinsic toxin HokB.

The site2-protease (S2P) family of intramembrane proteases (IMPs) is conserved in all kingdoms of life and cleaves transmembrane proteins within the membrane to regulate and maintain various cellular activities. RseP, an Escherichia coli S2P peptidase, is involved in the regulation of gene expression through the regulated cleavage of the two target membrane proteins (RseA and FecR) and in membrane quality control through the proteolytic elimination of remnant signal peptides. RseP is expected to have additional substrates and to be involved in other cellular processes. Recent studies have shown that cells express small membrane proteins (SMPs; single-spanning membrane proteins of approximately 50-100 amino acid residues) with crucial cellular functions. However, little is known about their metabolism, which affects their functions. This study investigated the possible RseP-catalyzed cleavage of E. coli SMPs based on the apparent similarity of the sizes and structures of SMPs to those of remnant signal peptides. We screened SMPs cleaved by RseP in vivo and in vitro and identified 14 SMPs, including HokB, an endogenous toxin that induces persister formation, as potential substrates. We demonstrated that RseP suppresses the cytotoxicity and biological functions of HokB. The identification of several SMPs as novel potential substrates of RseP provides a clue to a comprehensive understanding of the cellular roles of RseP and other S2P peptidases and highlights a novel aspect of the regulation of SMPs. IMPORTANCE Membrane proteins play an important role in cell activity and survival. Thus, understanding their dynamics, including proteolytic degradation, is crucial. E. coli RseP, an S2P family intramembrane protease, cleaves membrane proteins to regulate gene expression in response to environmental changes and to maintain membrane quality. To identify novel substrates of RseP, we screened small membrane proteins (SMPs), a group of proteins that have recently been shown to have diverse cellular functions, and identified 14 potential substrates. We also showed that RseP suppresses the cytotoxicity of the intrinsic toxin, HokB, an SMP that has been reported to induce persister cell formation, by degrading it. These findings provide new insights into the cellular roles of S2P peptidases and the functional regulation of SMPs.

Analyzing protein intermediate interactions in living E. coli cells using site-specific photo-crosslinking combined with chemical crosslinking.

Information on protein-protein interactions is crucial in understanding protein-mediated cellular processes; however, analyzing transient and unstable interactions in living cells is challenging. Here, we present a protocol capturing the interaction between an assembly intermediate form of a bacterial outer membrane protein and ß-barrel assembly machinery complex components. We describe steps for expression of a protein target, chemical crosslinking combined with in vivo photo-crosslinking and crosslinking detection procedures including immunoblotting. This protocol can be adapted to analyze interprotein interactions in other processes. For complete details on the use and execution of this protocol, please refer to Miyazaki et al. (2021).1.

Effective Mechanical Thrombectomy for Posterior Circulation Ischemia Using Magnetic Resonance Imaging-based Arterial Structures.

To improve the success of mechanical thrombectomy, three-dimensional turbo spin-echo (3D-TSE) sequences on T2WI can be employed to estimate the vascular structure of the posterior circulation. In addition to the short imaging time of 3D-TSE T2WI (33 sec), it can visualize the outer diameter of the main cerebral artery, including the occluded vessels. However, to date, the efficacy of mechanical thrombectomy in the posterior circulation remains unclear, and safer and more efficient mechanical thrombectomy procedures are required. Assessment of the anatomical variations in the posterior circulation using 3D-TSE T2WI is valuable for access decisions, device selection, and safe device guidance and retrieval techniques to the target vessel. Herein, we present representative cases of basilar artery and posterior cerebral artery occlusions in our institute and describe the utility of preoperative 3D-TSE T2WI in these patients.

Symptomatic atherosclerotic plaque accompanied by carotid web.

Here, we describe a case of a 67-year-old man who was transferred to our hospital with complaints of sudden upper right limb weakness and ataxia. Scattered acute cerebral infarction was found in the watershed zone between the left anterior cerebral artery and the middle cerebral artery territories. A shelf-like structure at the origin of the left carotid artery and a vulnerable plaque distal to the lesion was found. Symptomatic atherosclerotic plaque with concomitant carotid web was diagnosed. Carotid endarterectomy resulted in good revascularization. Hemodynamic changes associated with vascular stenosis are involved in atherosclerosis. The current case is valuable and shows that carotid web can cause not only embolic infarction but also cerebral thrombosis due to atheroma formation in the carotid artery.

Inner membrane YfgM-PpiD heterodimer acts as a functional unit that associates with the SecY/E/G translocon and promotes protein translocation.

PpiD and YfgM are inner membrane proteins that are both composed of an N-terminal transmembrane segment and a C-terminal periplasmic domain. Escherichia coli YfgM and PpiD form a stable complex that interacts with the SecY/E/G (Sec) translocon, a channel that allows protein translocation across the cytoplasmic membrane. Although PpiD is known to function in protein translocation, the functional significance of PpiD-YfgM complex formation as well as the molecular mechanisms of PpiD-YfgM and PpiD/YfgM-Sec translocon interactions remain unclear. Here, we conducted genetic and biochemical studies using yfgM and ppiD mutants and demonstrated that a lack of YfgM caused partial PpiD degradation at its C-terminal region and hindered the membrane translocation of Vibrio protein export monitoring polypeptide (VemP), a Vibrio secretory protein, in both E. coli and Vibrio alginolyticus. While ppiD disruption also impaired VemP translocation, we found that the yfgM and ppiD double deletion exhibited no additive or synergistic effects. Together, these results strongly suggest that both PpiD and YfgM are required for efficient VemP translocation. Furthermore, our site-directed in vivo photocrosslinking analysis revealed that the tetratricopeptide repeat domain of YfgM and a conserved structural domain (NC domain) in PpiD interact with each other and that YfgM, like PpiD, directly interacts with the SecG translocon subunit. Crosslinking analysis also suggested that PpiD-YfgM complex formation is required for these proteins to interact with SecG. In summary, we propose that PpiD and YfgM form a functional unit that stimulates protein translocation by facilitating their proper interactions with the Sec translocon.

A Photo-Crosslinking Approach to Monitoring the Assembly of an LptD Intermediate with LptE in a Living Cell.

Elucidating the dynamic behavior of proteins in living cells is extremely important for understanding the physiological roles of biological processes. The site-specific in vivo photo-crosslinking approach using a photoreactive unnatural amino acid enables the analysis of protein interactions with high spatial resolution in vivo. Recently, by improving the photo-crosslinking technique, we developed the "PiXie" method for the analysis of dynamic interactions of newly synthesized proteins. Here, we describe the detailed protocols of the "PiXie" method and its application to the analysis of the assembly processes of the lipopolysaccharide translocon components, a ß-barrel outer membrane protein, LptD, and a lipoprotein, LptE.

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP.

Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including Escherichia coli RseP. Structure-based chemical modification and cross-linking experiments indicated that the RseP domains surrounding the active center undergo conformational changes to expose the substrate-binding site, suggesting that RseP has a gating mechanism to regulate substrate entry. Furthermore, mutational analysis suggests that a conserved electrostatic linkage between the transmembrane and peripheral membrane-associated domains mediates the conformational changes. In vivo cleavage assays also support that the substrate transmembrane helix is unwound by strand addition to the intramembrane ß sheet of RseP and is clamped by a conserved asparagine residue at the active center for efficient cleavage. This mechanism underlying the substrate binding, i.e., unwinding and clamping, appears common across distinct families of intramembrane proteases that cleave transmembrane segments.

Intramural Hematoma in Vertebrobasilar Dolichoectasia-Related Stroke: A Retrospective Analysis of Six Consecutive Patients.

BACKGROUND: The pathophysiology underlying vertebrobasilar dolichoectasia (VBD) is largely unknown. However, a few reports have demonstrated that acute intramural hematoma (IMH) in VBD is associated with stroke. We aimed to investigate the clinical and radiological features of IMH in VBD and the role of IMH in predicting rupture and patient outcomes. METHODS: We retrospectively reviewed the medical records of patients treated in 2 stroke centers between January 2012 and December 2021. Patients presenting with VBD and stroke were eligible for study inclusion. We excluded patients with stroke caused by arterial dissection or artery-to-artery embolism. IMH was defined as a crescent-shaped area of high signal density in the vessel wall on axial computed tomography in the absence of an intimal flap, double lumen, and pearl-and-string sign. RESULTS: Six patients were analyzed. All presented with symptoms of brainstem/cerebellar infarction without headache. Interobserver agreement for the presence or absence of IMH was excellent (100%). IMH was detected in 5 patients. The positive predictive value of IMH for rupture was 80% (95% confidence interval, 28%-99.5%). The median time from symptom onset to rupture was 2.5 days (range, 1.5-4). Median computed tomography values were significantly higher within the IMH than those in the lumen of the basilar artery (70 vs. 44.5 Hounsfield units; P = 0.008). The modified Rankin scale score on day 30 after onset was 5 in 1 patient and 6 in the remaining 5. CONCLUSIONS: IMH in patients with VBD presenting with brainstem/cerebellar infarction should be regarded as a sign associated with a high risk of rupture.

Cryo-EM structure of the entire FtsH-HflKC AAA protease complex.

The membrane-bound AAA protease FtsH is the key player controlling protein quality in bacteria. Two single-pass membrane proteins, HflK and HflC, interact with FtsH to modulate its proteolytic activity. Here, we present structure of the entire FtsH-HflKC complex, comprising 12 copies of both HflK and HflC, all of which interact reciprocally to form a cage, as well as four FtsH hexamers with periplasmic domains and transmembrane helices enclosed inside the cage and cytoplasmic domains situated at the base of the cage. FtsH K61/D62/S63 in the ß2-ß3 loop in the periplasmic domain directly interact with HflK, contributing to complex formation. Pull-down and in vivo enzymatic activity assays validate the importance of the interacting interface for FtsH-HflKC complex formation. Structural comparison with the substrate-bound human m-AAA protease AFG3L2 offers implications for the HflKC cage in modulating substrate access to FtsH. Together, our findings provide a better understanding of FtsH-type AAA protease holoenzyme assembly and regulation.

True superficial temporal artery aneurysm: A case after extracranial-intracranial bypass surgery and a systematic review.

Background: Nontraumatic true superficial temporal artery aneurysm (STAA) is rare, and its characteristics and pathogenesis are unclear. Methods: We report a case of STAA and performed a systematic review of PubMed, Scopus, and Web of Science using the keyword "superficial temporal artery aneurysm" to include studies on STAA reported through July 2022. We excluded studies on STAA associated with trauma, arterial dissection, infection, or vasculitis. Results: A 63-year-old woman who underwent left superficial temporal artery (STA)-middle cerebral artery bypass surgery 8 years previously was diagnosed with an aneurysm located at the left STA. The blood flow volume estimated by ultrasonography was higher in the left STA than in the contralateral counterpart (114 mL/min vs. 32 mL/min). She underwent clipping surgery to prevent aneurysmal rupture without sequela. The lesion was diagnosed as a true aneurysm by histology. The systematic review identified 63 cases (including the present case) of nontraumatic true STAA. The median age of the patients was 57 (interquartile range [IQR]: 41-70) years. Most (90.5%) cases were detected as a palpable mass. Aneurysmal rupture occurred in only 1 (1.6%) case, despite the large size of aneurysms (median size: 13 [IQR: 8-20] mm) and the high frequency (33.3%) of aneurysmal growth during observation. Most (93.7%) patients underwent surgical resection of STAA without sequela. Conclusion: Our findings suggest that the pathogenesis of true STAA is promoted by hemodynamic stress. The systematic review clarified patients' and aneurysmal characteristics and treatment outcomes, providing further insight into the pathogenesis of nontraumatic true STAA.

3D Turbo Spin-echo MRI-based Mechanical Thrombectomy at Middle Cerebral Artery Bifurcations.

We describe three cases with acute middle cerebral artery (MCA) occlusion. From the pre-operative MRI, including three-dimensional turbo spin-echo sequences using T1WI and T2WI, we assessed both thrombus configuration and arterial anatomy at the MCA bifurcations. For efficient endovascular thrombectomy, we identified the applied MCA segment 2 (M2) branch, in which the main thrombus was buried. Sufficient recanalization after a single pass was achieved and the patients made a marked recovery. Although mechanical thrombectomy for M2 occlusion has not been of proven benefit, the endovascular procedure based on three-dimensional turbo spin-echo imaging is useful for more complete thrombus removal at MCA bifurcations.

Edge-strand of BepA interacts with immature LptD on the ß-barrel assembly machine to direct it to on- and off-pathways.

The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier. Escherichia coli periplasmic Zn-metallopeptidase BepA contributes to the maintenance of OM integrity through its involvement in the biogenesis and degradation of LptD, a ß-barrel protein component of the lipopolysaccharide translocon. BepA either promotes the maturation of LptD when it is on the normal assembly pathway (on-pathway) or degrades it when its assembly is compromised (off-pathway). BepA performs these functions probably on the ß-barrel assembly machinery (BAM) complex. However, how BepA recognizes and directs an immature LptD to different pathways remains unclear. Here, we explored the interactions among BepA, LptD, and the BAM complex. We found that the interaction of the BepA edge-strand located adjacent to the active site with LptD was crucial not only for proteolysis but also, unexpectedly, for assembly promotion of LptD. Site-directed crosslinking analyses indicated that the unstructured N-terminal half of the ß-barrel-forming domain of an immature LptD contacts with the BepA edge-strand. Furthermore, the C-terminal region of the ß-barrel-forming domain of the BepA-bound LptD intermediate interacted with a 'seam' strand of BamA, suggesting that BepA recognized LptD assembling on the BAM complex. Our findings provide important insights into the functional mechanism of BepA.

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion.

Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab.

The Escherichia coli S2P intramembrane protease RseP regulates ferric citrate uptake by cleaving the sigma factor regulator FecR.

Escherichia coli RseP, a member of the site-2 protease family of intramembrane proteases, is involved in the activation of the σE extracytoplasmic stress response and elimination of signal peptides from the cytoplasmic membrane. However, whether RseP has additional cellular functions is unclear. In this study, we used mass spectrometry-based quantitative proteomic analysis to search for new substrates that might reveal unknown physiological roles for RseP. Our data showed that the levels of several Fec system proteins encoded by the fecABCDE operon (fec operon) were significantly decreased in an RseP-deficient strain. The Fec system is responsible for the uptake of ferric citrate, and the transcription of the fec operon is controlled by FecI, an alternative sigma factor, and its regulator FecR, a single-pass transmembrane protein. Assays with a fec operon expression reporter demonstrated that the proteolytic activity of RseP is essential for the ferric citrate-dependent upregulation of the fec operon. Analysis using the FecR protein and FecR-derived model proteins showed that FecR undergoes sequential processing at the membrane and that RseP participates in the last step of this sequential processing to generate the N-terminal cytoplasmic fragment of FecR that participates in the transcription of the fec operon with FecI. A shortened FecR construct was not dependent on RseP for activation, confirming this cleavage step is the essential and sufficient role of RseP. Our study unveiled that E. coli RseP performs the intramembrane proteolysis of FecR, a novel physiological role that is essential for regulating iron uptake by the ferric citrate transport system.

Cerebral Amyloidoma Accompanied by Sjögren's Syndrome: A Case Report and Literature Review.

We present a 69-year-old woman with colorectal cancer and a left frontal lobe tumor that was diagnosed as a cerebral amyloidoma after surgical resection. Further postoperative systemic evaluation revealed another amyloidoma in her hip as well as Sjögren's syndrome. Systemic amyloidosis was not present. To the best of our knowledge, this is the first case of cerebral amyloidoma presenting as one of the multiple localized amyloidomas accompanied by Sjögren's syndrome. We also present a systematic review of 65 cerebral amyloidoma cases reported in the literature over the past 40 years and discuss patient characteristics and pathological and imaging findings associated with prognosis.

Involvement of a Membrane-Bound Amphiphilic Helix in Substrate Discrimination and Binding by an Escherichia coli S2P Peptidase RseP.

Intramembrane proteases (IMPs) are a unique class of proteases that catalyze the proteolysis within the membrane and regulate diverse cellular processes in various organisms. RseP, an Escherichia coli site-2 protease (S2P) family IMP, is involved in the regulation of an extracytoplasmic stress response through the cleavage of membrane-spanning anti-stress-response transcription factor (anti-σE) protein RseA. Extracytoplasmic stresses trigger a sequential cleavage of RseA, in which first DegS cleaves off its periplasmic domain, and RseP catalyzes the second cleavage of RseA. The two tandem-arranged periplasmic PDZ (PDZ tandem) domains of RseP serve as a size-exclusion filter which prevents the access of an intact RseA into the active site of RseP IMP domain. However, RseP's substrate recognition mechanism is not fully understood. Here, we found that a periplasmic region of RseP, located downstream of the PDZ tandem, contains a segment (named H1) predicted to form an amphiphilic helix. Bacterial S2P homologs with various numbers of PDZ domains have a similar amphiphilic helix in the corresponding region. We demonstrated that the H1 segment forms a partially membrane-embedded amphiphilic helix on the periplasmic surface of the membrane. Systematic and random mutagenesis analyses revealed that the H1 helix is important for the stability and proteolytic function of RseP and that mutations in the H1 segment can affect the PDZ-mediated substrate discrimination. Cross-linking experiments suggested that H1 directly interacts with the DegS-cleaved form of RseA. We propose that H1 acts as an adaptor required for proper arrangement of the PDZ tandem domain to perform its filter function and for substrate positioning for its efficient cleavage.

Fine interaction profiling of VemP and mechanisms responsible for its translocation-coupled arrest-cancelation.

Bacterial cells utilize monitoring substrates, which undergo force-sensitive translation elongation arrest, to feedback-regulate a Sec-related gene. Vibrio alginolyticus VemP controls the expression of SecD/F that stimulates a late step of translocation by undergoing export-regulated elongation arrest. Here, we attempted at delineating the pathway of the VemP nascent-chain interaction with Sec-related factors, and identified the signal recognition particle (SRP) and PpiD (a membrane-anchored periplasmic chaperone) in addition to other translocon components and a ribosomal protein as interacting partners. Our results showed that SRP is required for the membrane-targeting of VemP, whereas PpiD acts cooperatively with SecD/F in the translocation and arrest-cancelation of VemP. We also identified the conserved Arg-85 residue of VemP as a crucial element that confers PpiD-dependence to VemP and plays an essential role in the regulated arrest-cancelation. We propose a scheme of the arrest-cancelation processes of VemP, which likely monitors late steps in the protein translocation pathway.