Organelle-specific isoenzymes of plant V-ATPase as revealed by in vivo-FRET analysis

Background

The ability to specifically label proteins within living cells can provide information about their dynamics and function. To study a membrane protein, we fused a multi-functional reporter protein, HaloTag^®, to the extracellular domain of a truncated integrin.

Results

Using the HaloTag technology, we could study the localization, trafficking and processing of an integrin-HaloTag fusion, which we showed had cellular dynamics consistent with native integrins. By labeling live cells with different fluorescent impermeable and permeable ligands, we showed spatial separation of plasma membrane and internal pools of the integrin-HaloTag fusion, and followed these protein pools over time to study bi-directional trafficking. In addition to combining the HaloTag reporter protein with different fluorophores, we also employed an affinity tag to achieve cell capture.

Conclusion

The HaloTag technology was used successfully to study expression, trafficking, spatial separation and real-time translocation of an integrin-HaloTag fusion, thereby demonstrating that this technology can be a powerful tool to investigate membrane protein biology in live cells.     

Architecture of the ribosome-channel complex derived from native membranes

The mammalian Sec61 complex forms a protein translocation channel whose function depends upon its interaction with the ribosome and with membrane proteins of the endoplasmic reticulum (ER). To study these interactions, we determined structures of "native" ribosome-channel complexes derived from ER membranes. We find that the ribosome is linked to the channel by seven connections, but the junction may still provide a path for domains of nascent membrane proteins to move into the cytoplasm. In addition, the native channel is significantly larger than a channel formed by the Sec61 complex, due to the presence of a second membrane protein. We identified this component as TRAP, the translocon-associated protein complex. TRAP interacts with Sec61 through its transmembrane domain and has a prominent lumenal domain. The presence of TRAP in the native channel indicates that it may play a general role in translocation. Crystal structures of two Sec61 homologues were used to model the channel. This analysis indicates that there are four Sec61 complexes and two TRAP molecules in each native channel. Thus, we suggest that a single Sec61 complex may form a conduit for translocating polypeptides, while three copies of Sec61 play a structural role or recruit accessory factors such as TRAP.     

内质网膜上的转运使者——易位子相关蛋白（TRAP）

内质网膜蛋白在参与信号序列的识别、新生肽链的修饰、转运通道的形成等生理过程中发挥重要作用.易位子相关蛋白（translocon-associated protein, TRAP）是广泛存在于高等真核生物中的一种膜蛋白,其作为信号序列的受体蛋白位于内质网膜上.该蛋白能选择性地识别信号序列,并与Sec61相互作用形成一个以Sec61为核心、TRAP侧向延伸的椭圆状转运通道,从而靶向新生肽链进入内质网腔.近来研究发现,TRAP与蛋白质构象病、神经退行性疾病、肿瘤转移等疾病的发病机制有关.本文将对TRAP各个亚基的最新研究及其功能作一综述.     

Functional characterization of the trans-membrane domain interactions of the Sec61 protein translocation complex beta-subunit

Background  

In eukaryotic cells co- and post-translational protein translocation is mediated by the trimeric Sec61 complex. Currently, the role of the Sec61 complex β-subunit in protein translocation is poorly understood. We have shown previously that in Saccharomyces cerevisiae the trans-membrane domain alone is sufficient for the function of the β-subunit Sbh1p in co-translational protein translocation. In addition, Sbh1p co-purifies not only with the protein translocation channel subunits Sec61p and Sss1p, but also with the reticulon family protein Rtn1p.     

The ribosome quality control pathway can access nascent polypeptides stalled at the Sec61 translocon

Cytosolic ribosomes that stall during translation are split into subunits, and nascent polypeptides trapped in the 60S subunit are ubiquitinated by the ribosome quality control (RQC) pathway. Whether the RQC pathway can also target stalls during cotranslational translocation into the ER is not known. Here we report that listerin and NEMF, core RQC components, are bound to translocon-engaged 60S subunits on native ER membranes. RQC recruitment to the ER in cultured cells is stimulated by translation stalling. Biochemical analyses demonstrated that translocon-targeted nascent polypeptides that subsequently stall are polyubiquitinated in 60S complexes. Ubiquitination at the translocon requires cytosolic exposure of the polypeptide at the ribosome–Sec61 junction. This exposure can result from either failed insertion into the Sec61 channel or partial backsliding of translocating nascent chains. Only Sec61-engaged nascent chains early in their biogenesis were relatively refractory to ubiquitination. Modeling based on recent 60S–RQC and 80S–Sec61 structures suggests that the E3 ligase listerin accesses nascent polypeptides via a gap in the ribosome–translocon junction near the Sec61 lateral gate. Thus the RQC pathway can target stalled translocation intermediates for degradation from the Sec61 channel.     

The β Subunit of the Sec61 Complex Facilitates Cotranslational Protein Transport and Interacts with the Signal Peptidase during Translocation

The Sec61 complex is the central component of the protein translocation apparatus of the ER membrane. We have addressed the role of the β subunit (Sec61β) during cotranslational protein translocation. With a reconstituted system, we show that a Sec61 complex lacking Sec61β is essentially inactive when elongation and membrane targeting of a nascent chain occur at the same time. The translocation process is perturbed at a step where the nascent chain would be inserted into the translocation channel. However, if sufficient time is given for the interaction of the nascent polypeptide with the mutant Sec61 complex, translocation is almost normal. Thus Sec61β kinetically facilitates cotranslational translocation, but is not essential for it.

Using chemical cross-linking we show that Sec61β not only interacts with subunits of the Sec61 complex but also with the 25-kD subunit of the signal peptidase complex (SPC25), thus demonstrating for the first time a tight interaction between the SPC and the Sec61 complex. Interestingly, the cross-links between Sec61β and SPC25 and between Sec61β and Sec61α depend on the presence of membrane-bound ribosomes, suggesting that these interactions are induced when translocation is initiated. We propose that the SPC is transiently recruited to the translocation site, thus enhancing its activity.

     

The Ribosome-Sec61 Translocon Complex Forms a Cytosolically Restricted Environment for Early Polytopic Membrane Protein Folding

Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-translocon complex (RTC) through iterative cycles of translocation initiation and termination. It remains unknown, however, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to translocon components. To address this question, we used cysteine scanning mutagenesis to monitor aqueous accessibility of stalled translation intermediates to determine when, during biogenesis, hydrophilic peptide loops of the aquaporin-4 (AQP4) water channel are delivered to cytosolic and lumenal compartments. Results showed that following ribosome docking on the ER membrane, the nascent polypeptide was shielded from the cytosol as it emerged from the ribosome exit tunnel. Extracellular loops followed a well defined path through the ribosome, the ribosome translocon junction, the Sec61-translocon pore, and into the ER lumen coincident with chain elongation. In contrast, intracellular loops (ICLs) and C-terminalresidues exited the ribosome into a cytosolically shielded environment and remained inaccessible to both cytosolic and lumenal compartments until translation was terminated. Shielding of ICL1 and ICL2, but not the C terminus, became resistant to maneuvers that disrupt electrostatic ribosome interactions. Thus, the early folding landscape of polytopic proteins is shaped by a spatially restricted environment localized within the assembled ribosome translocon complex.     

The transmembrane domain is sufficient for Sbh1p function, its association with the Sec61 complex, and interaction with Rtn1p

The Sec61 protein translocation complex in the endoplasmic reticulum (ER) membrane is composed of three subunits. The alpha-subunit, called Sec61p in yeast, is a multispanning membrane protein that forms the protein conducting channel. The functions of the smaller, carboxyl-terminally tail-anchored beta subunit Sbh1p, its close homologue Sbh2p, and the gamma subunit Sss1p are not well understood. Here we show that co-translational protein translocation into the ER is reduced in sbh1Delta sbh2Delta cells, whereas there is a limited reduction of post-translational translocation and no effect on export of a mutant form of alpha-factor precursor for ER-associated degradation in the cytosol. The translocation defect and the temperature-sensitive growth phenotype of sbh1Delta sbh2Delta cells were rescued by expression of the transmembrane domain of Sbh1p alone, and the Sbh1p transmembrane domain was sufficient for coimmunoprecipitation with Sec61p and Sss1p. Furthermore, we show that Sbh1p co-precipitates with the ER transmembrane protein Rtn1p. Sbh1p-Rtn1p complexes do not appear to contain Sss1p and Sec61p. Our results define the transmembrane domain as the minimal functional domain of the Sec61beta homologue Sbh1p in ER translocation, identify a novel interaction partner for Shb1p, and imply that Sbh1p has additional functions that are not directly linked to protein translocation in association with the Sec61 complex.     

Sss1p Is Required to Complete Protein Translocon Activation

Protein translocation across the endoplasmic reticulum membrane occurs at the Sec61 translocon. This has two essential subunits, the channel-forming multispanning membrane protein Sec61p/Sec61α and the tail-anchored Sss1p/Sec61γ, which has been proposed to “clamp” the channel. We have analyzed the function of Sss1p using a series of domain mutants and found that both the cytosolic and transmembrane clamp domains of Sss1p are essential for protein translocation. Our data reveal that the cytosolic domain is required for Sec61p interaction but that the transmembrane clamp domain is required to complete activation of the translocon after precursor targeting to Sec61p.     

Coordination of N-glycosylation and protein translocation across the endoplasmic reticulum membrane by Sss1 protein

Secretory proteins are translocated across the endoplasmic reticulum (ER) membrane through a channel formed by three proteins, namely Sec61p, Sbh1p, and Sss1p (Johnson, A. E., and van Waes, M. A. (1999) Annu. Rev. Cell Dev. Biol. 15, 799-842). Sec61p and Sss1p are essential for translocation (Esnault, Y., Blondel, M. O., Deshaies, R. J., Schekman, R., and Kepes, F. (1993) EMBO J. 12, 4083-4093). Sec61p is a polytopic membrane protein that lines the protein translocation channel. The role of Sss1p is unknown. During import into the ER through the Sec61p channel, many proteins are N-glycosylated before translocation is completed. In addition, both the Sec61 channel and oligosaccharyl transferase (OST) copurify with ribosomes from rough ER, suggesting that OST is located in close proximity to the Sec61 channel (Gorlich, D., Prehn, S., Hartmann, E., Kalies, K.-U., and Rapoport, T. A. (1992) Cell 71, 489-503 and Wang, L., and Dobberstein, B. (1999) FEBS Lett. 457, 316-322). Here, we demonstrate a direct interaction between Sss1p and a subunit of OST, Wbp1p, using the split-ubiquitin system and co-immunoprecipitation. We generated mutants in the cytoplasmic domain of Sss1p that disturb the interaction with OST and are viable but display a translocation defect specific for proteins with glycosylation acceptor sites. Our data suggest that Sss1p coordinates translocation across the ER membrane and N-linked glycosylation of secretory proteins.     

In vitro binding of ribosomes to the beta subunit of the Sec61p protein translocation complex

The Sec61p complex forms the core element of the protein translocation complex (translocon) in the rough endoplasmic reticulum (rough ER) membrane. Translating or nontranslating ribosomes bind with high affinity to ER membranes that have been stripped of ribosomes or to liposomes containing purified Sec61p. Here we present evidence that the beta subunit of the complex (Sec61beta) makes contact with nontranslating ribosomes. A fusion protein containing the Sec61beta cytoplasmic domain (Sec61beta(c)) prevents the binding of ribosomes to stripped ER-derived membranes and also binds to ribosomes directly with an affinity close to the affinity of ribosomes for stripped ER-derived membranes. The ribosome binding activity of Sec61beta(c), like that of native ER membranes, is sensitive to high salt concentrations and is not based on an unspecific charge-dependent interaction of the relatively basic Sec61beta(c) domain with ribosomal RNA. Like stripped ER membranes, the Sec61beta(c) sequence binds to large ribosomal subunits in preference over small subunits. Previous studies have shown that Sec61beta is inessential for ribosome binding and protein translocation, but translocation is impaired by the absence of Sec61beta, and it has been proposed that Sec61beta assists in the insertion of nascent proteins into the translocation pore. Our results suggest a physical interaction of the ribosome itself with Sec61beta; this may normally occur alongside interactions between the ribosome and other elements of Sec61p, or it may represent one stage in a temporal sequence of binding.     

Impaired cortical processing of inspiratory loads in children with chronic respiratory defects

Background

We tested the hypothesis that maternal interleukin-1β (IL-1β) pretreatment and induction of fetal cortisol synthesis activates MAP kinases and thereby affects lung fluid absorption in preterm guinea pigs.

Methods

IL-1β was administered subcutaneously daily to timed-pregnant guinea pigs for three days. Fetuses were obtained by abdominal hysterotomy and instilled with isosmolar 5% albumin into the lungs and lung fluid movement was measured over 1 h by mass balance. MAP kinase expression was measured by western blot.

Results

Lung fluid absorption was induced at 61 days (D) gestation and stimulated at 68D gestation by IL-1β. Maternal IL-1β pretreatment upregulated ERK and upstream MEK expression at both 61 and 68D gestation, albeit being much more pronounced at 61D gestation. U0126 instillation completely blocked IL-1β-induced lung fluid absorption as well as IL-1β-induced/stimulated ERK expression. Cortisol synthesis inhibition by metyrapone attenuated ERK expression and lung fluid absorption in IL-1β-pretreated fetal lungs. JNK expression after maternal IL-1β pretreatment remained unaffected at either gestation age.

Conclusion

These data implicate the ERK MAP kinase pathway as being important for IL-1β induction/stimulation of lung fluid absorption in fetal guinea pigs.     

The protein translocation channel binds proteasomes to the endoplasmic reticulum membrane

Misfolded secretory proteins are transported across the endoplasmic reticulum (ER) membrane into the cytosol for degradation by proteasomes. A large fraction of proteasomes in a cell is associated with the ER membrane. We show here that binding of proteasomes to ER membranes is salt sensitive, ATP dependent, and mediated by the 19S regulatory particle. The base of the 19S particle, which contains six AAA-ATPases, binds to microsomal membranes with high affinity, whereas the 19S lid complex binds weakly. We demonstrate that ribosomes and proteasomes compete for binding to the ER membrane and have similar affinities for their receptor. Ribosomes bind to the protein conducting channel formed by the Sec61 complex in the ER membrane. We co-precipitated subunits of the Sec61 complex with ER-associated proteasome 19S particles, and found that proteoliposomes containing only the Sec61 complex retained proteasome binding activity. Collectively, our data suggest that the Sec61 channel is a principal proteasome receptor in the ER membrane.     

CK2 phosphorylation of human Sec63 regulates its interaction with Sec62

Background

Protein kinase CK2 is a pleiotropic enzyme which is ubiquitously expressed in eukaryotic cells. Several years ago CK2 was found to be associated with the mammalian endoplasmic reticulum. So far nothing is known about the function of CK2 at the ER.

Methods

CK2 phosphorylation sites in the polypeptide chain of Sec63 were mapped using deletion mutants and a peptide library. Binding of Sec63 to CK2 and to Sec62 was analyzed by pull-down assays and by co-immunoprecipitation

Results

Sec63 was identified as a novel substrate and binding partner of protein kinase CK2.We identified serine 574, serine 576 and serine 748 as CK2 phosphorylation sites. Phosphorylation of Sec63 by CK2 enhanced its binding to Sec62.

Conclusions

Protein kinase CK2 phosphorylation of Sec63 leads to an enhanced binding of Sec63 to Sec62. This complex formation is a prerequisite for a functional ER protein translocon.

General significance

Thus, our present data indicate a regulatory role of CK2 in the ER protein translocation.     

Peroxisome Formation Requires the Endoplasmic Reticulum Channel Protein Sec61

In peroxisome formation, models of near‐autonomous peroxisome biogenesis with membrane protein integration directly from the cytosol into the peroxisomal membrane are in direct conflict with models whereby peroxisomes bud from the endoplasmic reticulum and receive their membrane proteins through a branch of the secretory pathway. We therefore reinvestigated the role of the Sec 61 complex, the protein‐conducting channel of the endoplasmic reticulum (ER) in peroxisome formation. We found that depletion or partial inactivation of Sec 61 in yeast disables peroxisome formation. The ER entry of the early peroxisomal membrane protein Pex 3 engineered with a glycosylation tag is reduced in sec61 mutant cells. Moreover, we were able to reconstitute Pex 3 import into ER membranes in vitro, and we identified a variant of a signal anchor sequence for ER translocation at the Pex 3 N‐terminus. Our findings are consistent with a Sec 61 requirement for peroxisome formation and a fundamental role of the ER in peroxisome biogenesis.     

Loss of Specific Chaperones Involved in Membrane Glycoprotein Biosynthesis during the Maturation of Human Erythroid Progenitor Cells

The production of erythrocytes requires the massive synthesis of red cell-specific proteins including hemoglobin, cytoskeletal proteins, as well as membrane glycoproteins glycophorin A (GPA) and anion exchanger 1 (AE1). We found that during the terminal differentiation of human CD34⁺ erythroid progenitor cells in culture, key components of the endoplasmic reticulum (ER) protein translocation (Sec61α), glycosylation (OST48), and protein folding machinery, chaperones BiP, calreticulin (CRT), and Hsp90 were maintained to allow efficient red cell glycoprotein biosynthesis. Unexpected was the loss of calnexin (CNX), an ER glycoprotein chaperone, and ERp57, a protein-disulfide isomerase, as well as a major decrease of the cytosolic chaperones, Hsc70 and Hsp70, components normally involved in membrane glycoprotein folding and quality control. AE1 can traffic to the cell surface in mouse embryonic fibroblasts completely deficient in CNX or CRT, whereas disruption of the CNX/CRT-glycoprotein interactions in human K562 cells using castanospermine did not affect the cell-surface levels of endogenous GPA or expressed AE1. These results demonstrate that CNX and ERp57 are not required for major glycoprotein biosynthesis during red cell development, in contrast to their role in glycoprotein folding and quality control in other cells.The production of red blood cells involves the terminal differentiation of hematopoietic stem cells in the bone marrow followed by release into the peripheral blood (1, 2). Red blood cells remain in circulation for ∼120 days and require the prior production of abundant red cell-specific proteins including hemoglobin, cytoskeletal proteins, and membrane glycoproteins such as anion exchanger 1 (AE1)³ and glycophorin A (GPA). During differentiation, erythroid progenitor cells undergo extensive remodeling of their cytoskeleton and loss of nuclei and other organelles like the endoplasmic reticulum (ER). AE1 and GPA are known to be synthesized late in differentiation when these key cellular components are lost (3). The efficient biosynthesis of these red cell membrane glycoproteins, however, is expected to require robust ER assembly machinery involving protein translocation, N-glycosylation, and protein folding chaperones.The proper folding of membrane glycoproteins engages the quality control function of cytosolic and ER chaperone proteins (4, 5). Newly synthesized proteins undergo cycles of binding and release with chaperones, minimizing aggregation and facilitating folding. Chaperones also play a role in the retention and degradation of misfolded proteins and in apoptosis (6-8). The membrane-bound ER chaperone calnexin (CNX) and its luminal paralog calreticulin (CRT) interact with folding intermediates via their lectin and protein binding domains, thereby preventing aggregation (9). A wide variety of glycoprotein substrates have been identified, with some binding to one or both chaperones, and both have been shown to be vital in the prevention of aggregation and proper maturation of membrane glycoproteins (9, 10). Disruption of interactions with CNX and CRT can allow misfolded membrane glycoproteins to escape the ER and traffic to the plasma membrane (9).In the present study, we examined the integrity of the ER protein translocation, N-glycosylation, and quality control machinery during the differentiation of human CD34⁺ erythroid cells in culture. We found that specific components of the protein quality control system were completely lost (CNX and ERp57) or diminished (Hsc70 and Hsp70) before the production of the major glycoproteins, AE1 and GPA, was completed. Components of the protein translocation (Sec61α) and N-glycosylation machinery (OST48) were, however, maintained. Chaperones that play other roles in erythrocyte maturation and survival (CRT, BiP, and Hsp90) were also retained (11). AE1 was found to traffic efficiently to the plasma membrane in mouse embryonic fibroblasts completely lacking the ER chaperone CNX or CRT. Furthermore, disruption of CNX/CRT-glycoprotein interactions in human K562 cells did not affect the cell-surface expression of GPA or AE1. These results demonstrate that CNX and ERp57 are not required for the efficient synthesis and folding of red cell membrane glycoproteins during terminal erythropoiesis. The lack of engagement with the quality control and disulfide folding machinery may allow the more rapid production of red cell glycoproteins late in differentiation, sacrificing quality for quantity.     

The structure of ribosome-channel complexes engaged in protein translocation

Cotranslational translocation of proteins requires ribosome binding to the Sec61p channel at the endoplasmic reticulum (ER) membrane. We have used electron cryomicroscopy to determine the structures of ribosome-channel complexes in the absence or presence of translocating polypeptide chains. Surprisingly, the structures are similar and contain 3-4 connections between the ribosome and channel that leave a lateral opening into the cytosol. Therefore, the ribosome-channel junction may allow the direct transfer of polypeptides into the channel and provide a path for the egress of some nascent chains into the cytosol. Moreover, complexes solubilized from mammalian ER membranes contain an additional membrane protein that has a large, lumenal protrusion and is intercalated into the wall of the Sec61p channel. Thus, the native channel contains a component that is not essential for translocation.     

Estradiol effects on the dopamine transporter – protein levels, subcellular location, and function

Background

The effects of estrogens on dopamine (DA) transport may have important implications for the increased incidence of neurological disorders in women during life stages when hormonal fluctuations are prevalent, e.g. during menarche, reproductive cycling, pregnancy, and peri-menopause.

Results

The activity of the DA transporter (DAT) was measured by the specific uptake of ³H-DA. We found that low concentrations (10^-14 to 10^-8 M) of 17β-estradiol (E₂) inhibit uptake via the DAT in PC12 cells over 30 minutes, with significant inhibition taking place due to E₂ exposure during only the last five minutes of the uptake period. Such rapid action suggests a non-genomic, membrane-initiated estrogenic response mechanism. DAT and estrogen receptor-α (ERα) were elevated in cell extracts by a 20 ng/ml 2 day NGFβ treatment, while ERβ was not. DAT, ERα and ERβ were also detectable on the plasma membrane of unpermeabilized cells by immunocytochemical staining and by a fixed cell, quantitative antibody (Ab)-based plate assay. In addition, PC12 cells contained RNA coding for the alternative membrane ER GPR30; therefore, all 3 ER subtypes are candidates for mediating the rapid nongenomic actions of E₂. At cell densities above 15,000 cells per well, the E₂-induced inhibition of transport was reversed. Uptake activity oscillated with time after a 10 nM E₂ treatment; in a slower room temperature assay, inhibition peaked at 9 min, while uptake activity increased at 3 and 20–30 min. Using an Ab recognizing the second extracellular loop of DAT (accessible only on the outside of unpermeabilized cells), our immunoassay measured membrane vs. intracellular/nonvesicular DAT; both were found to decline over a 5–60 min E₂ treatment, though immunoblot analyses demonstrated no total cellular loss of protein.

Conclusion

Our results suggest that physiological levels of E₂ may act to sequester DAT in intracellular compartments where the transporter's second extramembrane loop is inaccessible (inside vesicles) and that rapid estrogenic actions on this differentiated neuronal cell type may be regulated via membrane ERs of several types.     

Degradation of subunits of the Sec61p complex, an integral component of the ER membrane, by the ubiquitin-proteasome pathway.

We have investigated the degradation of subunits of the trimeric Sec61p complex, a key component of the protein translocation apparatus of the ER membrane. A mutant form of Sec6lp and one of the two associated proteins (Sss1p) are selectively degraded, while the third constituent of the complex (Sbh1p) is stable. Our results demonstrate that the proteolysis of the multispanning membrane protein Sec61p is mediated by the ubiquitin-proteasome pathway, since it requires polyubiquitination, the presence of a membrane-bound (Ubc6) and a soluble (Ubc7) ubiquitin-conjugating enzyme and a functional proteasome. The process is proposed to be specific for unassembled Sec61p and Sss1p. Thus, our results suggest that one pathway of ER degradation of abnormal or unassembled membrane proteins is initiated at the cytoplasmic side of the ER.