Uncoupling translocation from translation: implications for transport of proteins across membranes

The segregation of secretory proteins into the cisternae of the endoplasmic reticulum (ER) is normally tightly coupled to their synthesis. This feature distinguishes their biogenesis from that of proteins targeted to many other organelles. In the examples presented, translocation across the ER membrane is dissociated from translation. Transport, which is normally cotranslational, may proceed in the absence of chain elongation. Moreover, translocation across the ER membrane does not proceed spontaneously since, even in the absence of protein synthesis, energy substrates are required for translocation. These conclusions have been extended to the cotranslational integration of newly synthesized transmembrane proteins.     

叶绿体运输蛋白的转运方式和运输机制研究

叶绿体的空间结构有6个区隔：外膜、膜间隙、内膜、基质、类囊体膜和类囊体腔。重点介绍了运输蛋白的跨外膜转运和跨内膜转运的运输机制,分别在叶绿体外膜易位子TOC和内膜易位子TIC的协助下实现跨膜转运。同时,介绍了分子伴侣的作用,它可提供前体蛋白运输的动力。     

Multiple mechanisms of protein insertion into and across membranes

Protein localization in cells is initiated by the binding of characteristic leader (signal) peptides to specific receptors on the membranes of mitochondria or endoplasmic reticulum or, in bacteria, to the plasma membrane. There are differences in the timing of protein synthesis and translocation into or across the bilayer and in the requirement for a transmembrane electrochemical potential. Comparisons of protein localization in these different membranes suggest underlying common mechanisms.     

Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum

Directionality in intracellular trafficking is essential to ensure the correct localization of proteins along the secretory pathway. Here, we found evidence for an active mechanism that prohibited back-fusion of de novo-generated vesicles with their donor compartment. Tip20p is a peripheral membrane protein implicated in consumption of COPI vesicles at the endoplasmic reticulum. However, a specific mutant of TIP20 did not interfere with COPII vesicle generation but allowed these vesicles to fuse back to the endoplasmic reticulum, a process that does not occur normally in the cell.     

Role of ER export signals in controlling surface potassium channel numbers

Little is known about the identity of endoplasmic reticulum (ER) export signals and how they are used to regulate the number of proteins on the cell surface. Here, we describe two ER export signals that profoundly altered the steady-state distribution of potassium channels and were required for channel localization to the plasma membrane. When transferred to other potassium channels or a G protein-coupled receptor, these ER export signals increased the number of functional proteins on the cell surface. Thus, ER export of membrane proteins is not necessarily limited by folding or assembly, but may be under the control of specific export signals.     

The function of a leader peptide in translocating charged amino acyl residues across a membrane

Insertion of bacteriophage coat proteins into the membrane of infected bacterial cells can be studied as a model system of protein translocation across membranes. The coat protein of the filamentous bacteriophage Pf3--which infects Pseudomonas aeruginosa--is 44 amino acids in length and has the same basic structure as the coat protein of bacteriophage M13, which infects Escherichia coli. However, unlike the Pf3 coat protein, the M13 coat protein is synthesized as a precursor (procoat) with a typical leader (signal) sequence, which is cleaved after membrane insertion. Nevertheless, when the gene encoding the Pf3 coat protein is expressed in E. coli, the protein is translocated across the membrane. Hybrid M13 and Pf3 coat proteins were constructed in an attempt to understand how the Pf3 coat protein is translocated without a leader sequence. These studies demonstrated that the extracellular regions of the proteins determined their cellular location. When three charged residues in this region were neutralized, the leader-free M13 coat protein was also inserted into the membrane. Differences in the water shell surrounding these residues may account for efficient membrane insertion of the protein without a leader sequence.     

The trans Golgi network: sorting at the exit site of the Golgi complex

The Golgi complex is a series of membrane compartments through which proteins destined for the plasma membrane, secretory vesicles, and lysosomes move sequentially. A model is proposed whereby these three different classes of proteins are sorted into different vesicles in the last Golgi compartment, the trans Golgi network. This compartment corresponds to a tubular reticulum on the trans side of the Golgi stack, previously called Golgi endoplasmic reticulum lysosomes (GERL).     

Parallel single-cell monitoring of receptor-triggered membrane translocation of a calcium-sensing protein module

Time courses of translocation of fluorescently conjugated proteins to the plasma membrane were simultaneously measured in thousands of individual rat basophilic leukemia cells. We found that the C2 domain---a calcium-sensing, lipid-binding protein module that is an essential regulator of protein kinase C and numerous other proteins---targeted proteins to the plasma membrane transiently if calcium was released from internal stores, and persistently in response to entry of extracellular calcium across the plasma membrane. The C2 domain translocation time courses of stimulated cells clustered into only two primary modes. Hence, the reversible recruitment of families of signaling proteins from one cellular compartment to another is a rapid bifurcation mechanism for inducing discrete states of cellular signaling networks.     

Role of a highly conserved bacterial protein in outer membrane protein assembly

After transport across the cytoplasmic membrane, bacterial outer membrane proteins are assembled into the outer membrane. Meningococcal Omp85 is a highly conserved protein in Gram-negative bacteria, and its homolog Toc75 is a component of the chloroplast protein-import machinery. Omp85 appeared to be essential for viability, and unassembled forms of various outer membrane proteins accumulated upon Omp85 depletion. Immunofluorescence microscopy revealed decreased surface exposure of outer membrane proteins, which was particularly apparent at the cell-division planes. Thus, Omp85 is likely to play a role in outer membrane protein assembly.     

Protein translocation across biological membranes

Subcellular compartments have unique protein compositions, yet protein synthesis only occurs in the cytosol and in mitochondria and chloroplasts. How do proteins get where they need to go? The first steps are targeting to an organelle and efficient translocation across its limiting membrane. Given that most transport systems are exquisitely substrate specific, how are diverse protein sequences recognized for translocation? Are they translocated as linear polypeptide chains or after folding? During translocation, how are diverse amino acyl side chains accommodated? What are the proteins and the lipid environment that catalyze transport and couple it to energy? How is translocation coordinated with protein synthesis and folding, and how are partially translocated transmembrane proteins released into the lipid bilayer? We review here the marked progress of the past 35 years and salient questions for future work. Subcellular compartments have unique protein compositions, yet protein synthesis only occurs in the cytosol and in mitochondria and chloroplasts. How do proteins get where they need to go? The first steps are targeting to an organelle and efficient translocation across its limiting membrane. Given that most transport systems are exquisitely substrate specific, how are diverse protein sequences recognized for translocation? Are they translocated as linear polypeptide chains or after folding? During translocation, how are diverse amino acyl side chains accommodated? What are the proteins and the lipid environment that catalyze transport and couple it to energy? How is translocation coordinated with protein synthesis and folding, and how are partially translocated transmembrane proteins released into the lipid bilayer? We review here the marked progress of the past 35 years and salient questions for future work.     

Membrane insertion of a potassium-channel voltage sensor

The mechanism of voltage gating in K+ channels is controversial. The paddle model posits that highly charged voltage-sensor domains move relatively freely across the lipid bilayer in response to membrane depolarization; competing models picture the charged S4 voltage-sensor helix as being shielded from lipid contact by other parts of the protein. We measured the apparent free energy of membrane insertion of a K+-channel S4 helix into the endoplasmic reticulum membrane and conclude that S4 is poised very near the threshold of efficient bilayer insertion. Our results suggest that the paddle model is not inconsistent with the high charge content of S4.     

Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1

Malfolded proteins in the endoplasmic reticulum (ER) induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs). Mammalian homologs of yeast IRE1, which activate chaperone genes in response to ER stress, also activated JNK, and IRE1alpha-/- fibroblasts were impaired in JNK activation by ER stress. The cytoplasmic part of IRE1 bound TRAF2, an adaptor protein that couples plasma membrane receptors to JNK activation. Dominant-negative TRAF2 inhibited activation of JNK by IRE1. Activation of JNK by endogenous signals initiated in the ER proceeds by a pathway similar to that initiated by cell surface receptors in response to extracellular signals.     

Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators

Rhizobial bacteria enter a symbiotic interaction with legumes, activating diverse responses in roots through the lipochito oligosaccharide signaling molecule Nod factor. Here, we show that NSP2 from Medicago truncatula encodes a GRAS protein essential for Nod-factor signaling. NSP2 functions downstream of Nod-factor-induced calcium spiking and a calcium/calmodulin-dependent protein kinase. We show that NSP2-GFP expressed from a constitutive promoter is localized to the endoplasmic reticulum/nuclear envelope and relocalizes to the nucleus after Nod-factor elicitation. This work provides evidence that a GRAS protein transduces calcium signals in plants and provides a possible regulator of Nod-factor-inducible gene expression.     

Requirement of GTP hydrolysis for dissociation of the signal recognition particle from its receptor.

The signal recognition particle (SRP) directs signal sequence specific targeting of ribosomes to the rough endoplasmic reticulum. Displacement of the SRP from the signal sequence of a nascent polypeptide is a guanosine triphosphate (GTP)-dependent reaction mediated by the membrane-bound SRP receptor. A nonhydrolyzable GTP analog can replace GTP in the signal sequence displacement reaction, but the SRP then fails to dissociate from the membrane. Complexes of the SRP with its receptor containing the nonhydrolyzable analog are incompetent for subsequent rounds of protein translocation. Thus, vectorial targeting of ribosomes to the endoplasmic reticulum is controlled by a GTP hydrolysis cycle that regulates the affinity between the SRP, signal sequences, and the SRP receptor.     

Chaperone activity of protein O-fucosyltransferase 1 promotes notch receptor folding

Notch proteins are receptors for a conserved signaling pathway that affects numerous cell fate decisions. We found that in Drosophila, Protein O-fucosyltransferase 1 (OFUT1), an enzyme that glycosylates epidermal growth factor-like domains of Notch, also has a distinct Notch chaperone activity. OFUT1 is an endoplasmic reticulum protein, and its localization was essential for function in vivo. OFUT1 could bind to Notch, was required for the trafficking of wild-type Notch out of the endoplasmic reticulum, and could partially rescue defects in secretion and ligand binding associated with Notch point mutations. This ability of OFUT1 to facilitate folding of Notch did not require its fucosyltransferase activity. Thus, a glycosyltransferase can bind its substrate in the endoplasmic reticulum to facilitate normal folding.     

Membrane proteins of the endoplasmic reticulum induce high-curvature tubules

The tubular structure of the endoplasmic reticulum (ER) appears to be generated by integral membrane proteins, the reticulons and a protein family consisting of DP1 in mammals and Yop1p in yeast. Here, individual members of these families were found to be sufficient to generate membrane tubules. When we purified yeast Yop1p and incorporated it into proteoliposomes, narrow tubules (approximately 15 to 17 nanometers in diameter) were generated. Tubule formation occurred with different lipids; required essentially only the central portion of the protein, including its two long hydrophobic segments; and was prevented by mutations that affected tubule formation in vivo. Tubules were also formed by reconstituted purified yeast Rtn1p. Tubules made in vitro were narrower than normal ER tubules, due to a higher concentration of tubule-inducing proteins. The shape and oligomerization of the "morphogenic" proteins could explain the formation of the tubular ER.     

Distinct modes of signal recognition particle interaction with the ribosome

Signal recognition particle (SRP), together with its receptor (SR), mediates the targeting of ribosome-nascent chain complexes to the endoplasmic reticulum. Using protein cross-linking, we detected distinct modes in the binding of SRP to the ribosome. During signal peptide recognition, SRP54 is positioned at the exit site close to ribosomal proteins L23a and L35. When SRP54 contacts SR, SRP54 is rearranged such that it is no longer close to L23a. This repositioning may allow the translocon to dock with the ribosome, leading to insertion of the signal peptide into the translocation channel.     

Proton motive force involved in protein transport across the outer membrane of Aeromonas salmonicida

Many Gram-negative bacteria export proteins to the exterior. Some of these proteins are first secreted into the periplasm and then cross the outer membrane in a separate step. The source of energy required for the translocation is unknown. Export of the extracellular protein proaerolysin from the periplasm through the outer membrane of Aeromonas salmonicida is inhibited by a proton ionophore and by low extracellular pH. One possible explanation of these results is that a proton gradient across the outer membrane is required for export.     

‘航花2号’的Δ~(12)脂肪酸脱氢酶(FAD2)基因的生物信息学分析

植物中的Δ12脂肪酸脱氢酶(FAD2)是油酸形成亚油酸的关键酶。通过NCBI和EXPASY 2大数据库,采用Signal P、TMHMM、Psort、Prot Param和Target P等分析程序对花生品种‘航花2号’及其它物种的19个FAD2蛋白序列进行分析,利用MEGA6.0软件比对序列及构建系统进化树阐明FAD2基因的系统发育关系,亲缘相近的FAD2蛋白聚在一起。预测了‘航花2号’和‘粤油13’的FAD2蛋白的分子质量、等电点、信号肽、跨膜和保守结构域等。结果表明:‘航花2号’FAD2蛋白的N端没有信号肽,预测定位在微体、细胞质、线粒体基质和叶绿体类囊膜中,而C端信号基序LKGL使得FAD2蛋白选择性地结合和嵌入内质网,植物的FAD2蛋白普遍有3个高度保守的组氨酸富集基序(HECGHH、HRRHH和H[A/C/T]HH)和3~5个跨膜结构,但分析结果显示,‘航花2号’的FAD2蛋白的H[A/C/T]HH的组氨酸基序是缺失的,而油酸转化为亚油酸的效率上并没有显著变化,为将来FAD2基因的基因工程操作提供一定的理论基础。