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.     

N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli

N-linked protein glycosylation is the most abundant posttranslation modification of secretory proteins in eukaryotes. A wide range of functions are attributed to glycan structures covalently linked to asparagine residues within the asparagine-X-serine/threonine consensus sequence (Asn-Xaa-Ser/Thr). We found an N-linked glycosylation system in the bacterium Campylobacter jejuni and demonstrate that a functional N-linked glycosylation pathway could be transferred into Escherichia coli. Although the bacterial N-glycan differs structurally from its eukaryotic counterparts, the cloning of a universal N-linked glycosylation cassette in E. coli opens up the possibility of engineering permutations of recombinant glycan structures for research and industrial applications.     

Export-mediated assembly of mycobacterial glycoproteins parallels eukaryotic pathways

Protein O-mannosylation is an essential and evolutionarily conserved post-translational modification among eukaryotes. This form of protein modification is also described in Mycobacterium tuberculosis; however, the mechanism of mannoprotein assembly remains unclear. Evaluation of differentially translocated chimeric proteins and mass spectrometry to monitor glycosylation demonstrated that specific translocation processes were required for protein O-mannosylation in M. tuberculosis. Additionally, Rv1002c, a M. tuberculosis membrane protein homolog of eukaryotic protein mannosyltransferases, was shown to catalyze the initial step of protein mannosylation. Thus, the process of protein mannosylation is conserved between M. tuberculosis and eukaryotic organisms.     

蛋白质分子的一生——蛋白质的品质管理(英文)

概述了蛋白质品质管理中涉及的分子伴侣、激发未折叠蛋白反应(unfolded protein response, UPR)和内质网相关性蛋白质降解途径(ER-associated degradation, ERAD)等的研究进展,并探讨了该领域存在的问题以及发展前景。指出蛋白质的生命过程经历生成、折叠、组装和降解,每个过程都有严格控制。内质网中,各种蛋白质合成、折叠并经修饰形成具有一定构象的功能性蛋白。其在内质网折叠受阻碍时,未折叠的蛋白聚集,激发 UPR,使一系列分子伴侣和蛋白质折叠所需修饰酶类表达上调,帮助其完成折叠和装配。如果这些蛋白仍不能正确折叠,则进入 ERAD 被降解。     

蛋白质的品质管理

概述了蛋白质品质管理中涉及的分子伴侣、激发未折叠蛋白反应(unfolded protein response,UPR)和内质网相关性蛋白质降解途径(ER-associated degradation,ERAD)等的研究进展,并探讨了该领域存在的问题以及发展前景。指出蛋白质的生命过程经历生成、折叠、组装和降解,每个过程都有严格控制。内质网中,各种蛋白质合成、折叠并经修饰形成具有一定构象的功能性蛋白。其在内质网折叠受阻碍时,未折叠的蛋白聚集,激发UPR,使一系列分子伴侣和蛋白质折叠所需修饰酶类表达上调,帮助其完成折叠和装配。如果这些蛋白仍不能正确折叠,则进入ERAD被降解。     

Ankyrin repeat proteins comprise a diverse family of bacterial type IV effectors

Specialized secretion systems are used by many bacteria to deliver effector proteins into host cells that can either mimic or disrupt the function of eukaryotic factors. We found that the intracellular pathogens Legionella pneumophila and Coxiella burnetii use a type IV secretion system to deliver into eukaryotic cells a large number of different bacterial proteins containing ankyrin repeat homology domains called Anks. The L. pneumophila AnkX protein prevented microtubule-dependent vesicular transport to interfere with fusion of the L. pneumophila-containing vacuole with late endosomes after infection of macrophages, which demonstrates that Ank proteins have effector functions important for bacterial infection of eukaryotic host cells.     

Simulations of the folding of a globular protein

Dynamic Monte Carlo simulations of the folding of a globular protein, apoplastocyanin, have been undertaken in the context of a new lattice model of proteins that includes both side chains and a-carbon backbone atoms and that can approximate native conformations at the level of 2 angstroms (root mean square) or better. Starting from random-coil unfolded states, the model apoplastocyanin was folded to a native conformation that is topologically similar to the real protein. The present simulations used a marginal propensity for local secondary structure consistent with but by no means enforcing the native conformation and a full hydrophobicity scale in which any nonbonded pair of side chains could interact. These molecules folded through a punctuated on-site mechanism of assembly where folding initiated at or near one of the turns ultimately found in the native conformation. Thus these simulations represent a partial solution to the globular-protein folding problem.     

Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1

Eukaryotic ribosomes are substantially larger and more complex than their bacterial counterparts. Although their core function is conserved, bacterial and eukaryotic protein synthesis differ considerably at the level of initiation. The eukaryotic small ribosomal subunit (40S) plays a central role in this process; it binds initiation factors that facilitate scanning of messenger RNAs and initiation of protein synthesis. We have determined the crystal structure of the Tetrahymena thermophila 40S ribosomal subunit in complex with eukaryotic initiation factor 1 (eIF1) at a resolution of 3.9 angstroms. The structure reveals the fold of the entire 18S ribosomal RNA and of all ribosomal proteins of the 40S subunit, and defines the interactions with eIF1. It provides insights into the eukaryotic-specific aspects of protein synthesis, including the function of eIF1 as well as signaling and regulation mediated by the ribosomal proteins RACK1 and rpS6e.     

Atomic-level characterization of the structural dynamics of proteins

Molecular dynamics (MD) simulations are widely used to study protein motions at an atomic level of detail, but they have been limited to time scales shorter than those of many biologically critical conformational changes. We examined two fundamental processes in protein dynamics--protein folding and conformational change within the folded state--by means of extremely long all-atom MD simulations conducted on a special-purpose machine. Equilibrium simulations of a WW protein domain captured multiple folding and unfolding events that consistently follow a well-defined folding pathway; separate simulations of the protein's constituent substructures shed light on possible determinants of this pathway. A 1-millisecond simulation of the folded protein BPTI reveals a small number of structurally distinct conformational states whose reversible interconversion is slower than local relaxations within those states by a factor of more than 1000.     

Mutants of bovine pancreatic trypsin inhibitor lacking cysteines 14 and 38 can fold properly

It is a generally accepted principle of biology that a protein's primary sequence is the main determinant of its tertiary structure. However, the mechanism by which a protein proceeds from an unfolded, disordered state to a folded, relatively well-ordered, native conformation is obscure. Studies have been initiated to examine the "genetics" of protein folding, with mutants of bovine pancreatic trypsin inhibitor (BPTI) being used to explore the nature of the specific intramolecular interactions that direct this process. Previous work with BPTI chemically modified at cysteines 14 and 38 indicated that transient disulfide bond formation by these residues contributed to efficient folding at 25 degrees C. In the present work, mutants of BPTI in which these cysteines were replaced by alanines or threonines were made and the mutant proteins were produced by a heterologous Escherichia coli expression system. At 25 degrees C in vitro, the refolding behavior of these mutants was characterized by a pronounced lag. However, when expressed at 37 degrees C in E. coli, or when refolded at 37 degrees or 52 degrees C in vitro, the mutant proteins folded readily into the native conformation, albeit at a rate somewhat slower than that exhibited by wild-type BPTI. These results indicate that, at physiological temperatures, BPTI lacking cysteines 14 and 38 can refold quantitatively.     

Setting the standards: quality control in the secretory pathway

A variety of quality control mechanisms operate in the endoplasmic reticulum and in downstream compartments of the secretory pathway to ensure the fidelity and regulation of protein expression during cell life and differentiation. As a rule, only proteins that pass a stringent selection process are transported to their target organelles and compartments. If proper maturation fails, the aberrant products are degraded. Quality control improves folding efficiency by retaining proteins in the special folding environment of the endoplasmic reticulum, and it prevents harmful effects that could be caused by the deployment of incompletely folded or assembled proteins.     

Stop-transfer regions do not halt translocation of proteins into chloroplasts

Protein targeting in eukaryotic cells is determined by several topogenic signals. Among these are stop-transfer regions, which halt translocation of proteins across the endoplasmic reticulum membrane. Two different stop-transfer regions were incorporated into precursors for a chloroplast protein, the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Both chimeric proteins were imported into chloroplasts and did not accumulate in the envelope membranes. Thus, the stop-transfer signals did not function during chloroplast protein import. These observations support the hypothesis that the mechanism for translocation of proteins across the chloroplast envelope is significantly different from that for translocation across the endoplasmic reticulum membrane.     

Human Mpp11 J protein: ribosome-tethered molecular chaperones are ubiquitous

The existence of specialized molecular chaperones that interact directly with ribosomes is well established in microorganisms. Such proteins bind polypeptides exiting the ribosomal tunnel and provide a physical link between translation and protein folding. We report that ribosome-associated molecular chaperones have been maintained throughout eukaryotic evolution, as illustrated by Mpp11, the human ortholog of the yeast ribosome-associated J protein Zuo. When expressed in yeast, Mpp11 partially substituted for Zuo by partnering with the multipurpose Hsp70 Ssa, the homolog of mammalian Hsc70. We propose that in metazoans, ribosome-associated Mpp11 recruits the multifunctional soluble Hsc70 to nascent polypeptide chains as they exit the ribosome.     

Structure of a Bag/Hsc70 complex: convergent functional evolution of Hsp70 nucleotide exchange factors

Bag (Bcl2-associated athanogene) domains occur in a class of cofactors of the eukaryotic chaperone 70-kilodalton heat shock protein (Hsp70) family. Binding of the Bag domain to the Hsp70 adenosine triphosphatase (ATPase) domain promotes adenosine 5'-triphosphate-dependent release of substrate from Hsp70 in vitro. In a 1.9 angstrom crystal structure of a complex with the ATPase of the 70-kilodalton heat shock cognate protein (Hsc70), the Bag domain forms a three-helix bundle, inducing a conformational switch in the ATPase that is incompatible with nucleotide binding. The same switch is observed in the bacterial Hsp70 homolog DnaK upon binding of the structurally unrelated nucleotide exchange factor GrpE. Thus, functional convergence has allowed proteins with different architectures to trigger a conserved conformational shift in Hsp70 that leads to nucleotide exchange.     

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.     

Protein native-state stabilization by placing aromatic side chains in N-glycosylated reverse turns

N-glycosylation of eukaryotic proteins helps them fold and traverse the cellular secretory pathway and can increase their stability, although the molecular basis for stabilization is poorly understood. Glycosylation of proteins at na?ve sites (ones that normally are not glycosylated) could be useful for therapeutic and research applications but currently results in unpredictable changes to protein stability. We show that placing a phenylalanine residue two or three positions before a glycosylated asparagine in distinct reverse turns facilitates stabilizing interactions between the aromatic side chain and the first N-acetylglucosamine of the glycan. Glycosylating this portable structural module, an enhanced aromatic sequon, in three different proteins stabilizes their native states by -0.7 to -2.0 kilocalories per mole and increases cellular glycosylation efficiency.     

Detecting and measuring cotranslational protein degradation in vivo

Nascent polypeptides emerging from the ribosome and not yet folded may at least transiently present degradation signals similar to those recognized by the ubiquitin system in misfolded proteins. The ubiquitin sandwich technique was used to detect and measure cotranslational protein degradation in living cells. More than 50 percent of nascent protein molecules bearing an amino-terminal degradation signal can be degraded cotranslationally, never reaching their mature size before their destruction by processive proteolysis. Thus, the folding of nascent proteins, including abnormal ones, may be in kinetic competition with pathways that target these proteins for degradation cotranslationally.     

Structure of the membrane protein FhaC: a member of the Omp85-TpsB transporter superfamily

In Gram-negative bacteria and eukaryotic organelles, beta-barrel proteins of the outer membrane protein 85-two-partner secretion B (Omp85-TpsB) superfamily are essential components of protein transport machineries. The TpsB transporter FhaC mediates the secretion of Bordetella pertussis filamentous hemagglutinin (FHA). We report the 3.15 A crystal structure of FhaC. The transporter comprises a 16-stranded beta barrel that is occluded by an N-terminal alpha helix and an extracellular loop and a periplasmic module composed of two aligned polypeptide-transport-associated (POTRA) domains. Functional data reveal that FHA binds to the POTRA 1 domain via its N-terminal domain and likely translocates the adhesin-repeated motifs in an extended hairpin conformation, with folding occurring at the cell surface. General features of the mechanism obtained here are likely to apply throughout the superfamily.     

Coordinated nonvectorial folding in a newly synthesized multidomain protein

The low-density lipoprotein receptor (LDL-R) is a typical example of a multidomain protein, for which in vivo folding is assumed to occur vectorially from the amino terminus to the carboxyl terminus. Using a pulse-chase approach in intact cells, we found instead that newly synthesized LDL-R molecules folded by way of "collapsed" intermediates that contained non-native disulfide bonds between distant cysteines. The most amino-terminal domain acquired its native conformation late in folding instead of during synthesis. Thus, productive LDL-R folding in a cell is not vectorial but is mostly posttranslational, and involves transient long-range non-native disulfide bonds that are isomerized into native short-range cysteine pairs.