Bernhard Dobberstein

Common Principles of Protein Translocation Across Membranes

Most major systems that transport proteins across a membrane share the following features: an amino-terminal transient signal sequence on the transported protein, a targeting system on the cis side of the membrane, a hetero-oligomeric transmembrane channel that is gated both across and within the plane of the membrane, a peripherally attached protein translocation motor that is powered by the hydrolysis of nucleoside triphosphate, and a protein folding system on the trans side of the membrane. These transport systems are divided into two families: export systems that export proteins out of the cytosol, and import systems that transport proteins into cytosol-like compartments.

MHC class II-associated invariant chain contains a sorting signal for endosomal compartments

The invariant chain (Ii) is a transmembrane protein that associates with the MHC class II molecules in the endoplasmic reticulum. Expression of Ii in MHC class II-negative CV1 cells showed that it acquired complex-type oligosaccharide side chains and was retained in endosomal compartments. To search for a sorting signal, we made progressive deletions from the cytoplasmic N-terminus of Ii. Deleting 11 amino acid residues resulted in a protein that was still sorted and retained in endosomal vesicles, whereas deletion of 15 or more amino acid residues resulted in a protein that became resident in the plasma membrane. Amino acids 12-15 are thus essential for intracellular transport to endosomal compartments. As Ii is intracellularly associated with the MHC class II molecules, it is proposed that Ii determines the intracellular transport route of these molecules.

Signal sequences: more than just greasy peptides

Export signal sequences target newly synthesized proteins to the endoplasmic reticulum of eukaryotic cells and the plasma membrane of bacteria. All signal sequences contain a hydrophobic core region, but, despite this, they show great variation in both overall length and amino acid sequence. Recently, it has become clear that this variation allows signal sequences to specify different modes of targeting and membrane insertion and even to perform functions after being cleaved from the parent protein. This review argues that signal sequences are not simply greasy peptides but sophisticated, multipurpose peptides containing a wealth of functional information.

The Protein-Conducting Channel in the Membrane of the Endoplasmic Reticulum Is Open Laterally toward the Lipid Bilayer

Lipids and proteins were found to contact a nascent type II membrane protein, as well as a nascent secretory protein, during their insertion into the membrane of the endoplasmic reticulum. This suggests that the protein-conducting channel is open laterally toward the lipid bilayer during an early stage of protein insertion. Contact to lipids was confined to the hydrophobic core region of the respective signal or signal anchor sequence. Thus, the nascent polypeptide is positioned in the translocation complex such that the signal or signal anchor sequence faces the lipid bilayer, whereas the hydrophilic, translocating portion is in proteinaceous environment.

Membrane insertion and oligomeric assembly of HLA-DR histocompatibility antigens

HLA-DR histocompatibility antigens are assembled in the endoplasmic reticulum. This assembly has been studied in vitro and in vivo. Three polypeptides are involved in forming the oligomeric structure of HLA-DR antigens, DR alpha chains (molecular weight 35,000), DR beta chains (molecular weight 29,000) and DR gamma chains (molecular weight 33,000). They are cotranslationally inserted into the membrane of the endoplasmic reticulum, and all span the membrane. The size of the cytoplasmic portion of DR alpha and DR beta is about 500- 1000 daltons, whereas that of the DR gamma chain is about 3000 daltons. Oligomeric assembly of DR alpha, DR beta and DR gamma chains occurs shortly after their synthesis in the endoplasmic reticulum. DR gamma chains are synthesized in excess of DR alpha and DR beta chains, and hence in the endoplasmic reticulum they are found either in a complex with DR alpha and DR beta or in a free form. Free DR gamma chains remain in the endoplasmic reticulum, whereas DR gamma chains present in the oligomeric complex with DR alpha and DR beta undergo intracellular transport. Their molecular weight increases during transport, probably because of the addition of complex sugars in the Golgi complex. This is followed by the detachment of DR gamma chains from the oligomeric complex and the appearance of DR alpha and DR beta chains on the cell surface. Whether any DR gamma chains appear on the cell surface is uncertain.

An alternative protein targeting pathway in Escherichia coli: studies on the role of FtsY

In Escherichia coli, a signal recognition particle (SRP) has been identified which binds specifically to the signal sequence of presecretory proteins and which appears to be essential for efficient translocation of a subset of proteins. In this study we have investigated the function of E. coli FtsY which shares sequence similarity with the alpha‐subunit of the eukaryotic SRP receptor (‘docking protein’) in the membrane of the endoplasmic reticulum. A strain was constructed which allows the conditional expression of FtsY. Depletion of FtsY is shown to cause the accumulation of the precursor form of beta‐lactamase, OmpF and ribose binding protein in vivo, whereas the processing of various other presecretory proteins is unaffected. Furthermore, FtsY‐depleted inverted cytoplasmic membrane vesicles are shown to be defective in the translocation of pre‐beta‐lactamase using an in vitro import assay. Subcellular localization studies revealed that FtsY is located in part at the cytoplasmic membrane with which it seems peripherally associated. These observations suggest that FtsY is the functional E. coli homolog of the mammalian SRP receptor.

Cell-free synthesis and membrane insertion of mouse H-2Dd histocompatibility antigen and β2-microglobulin

Abstract Messenger RNA from SL2 lymphoma cells was translated in a cell-free system in the presence of microsomal membranes. Mouse H-2D d histocompatibility antigen was correctly assembled in the microsomal membranes, and transmembrane insertion of the nascent chain was accompanied by glycosylation and cleavage of the signal sequence. H-2K d antigens, synthesized in vivo, comprised a transmembrane glycoprotein and an unglycosylated protein in the cytoplasm. The glycosylated forms of the H-2D d and H-2K d antigens were modified during intracellular transport from the endoplasmic reticulum to the cell surface. β 2 -Microglobulin was also synthesized in vitro, and transfer of this protein into microsomal vesicles was accompanied by cleavage of its signal sequence. In the endoplasmic reticulum, β 2 -microglobulin can bind to newly synthesized H-2 d glycoproteins. The mRNAs coding for β 2 -microglobulin and H-2D d antigen could be separated on aqueous sucrose gradients.

E. coli 4.5S RNA is part of a ribonucleoprotein particle that has properties related to signal recognition particle

E. coli 4.5S RNA and P48 have been shown to be homologous to SRP7S RNA and SRP54, respectively. Here we report that expression of human SRP7S in E. coli can suppress the lethality caused by depletion of 4.5S RNA. In E. coli, both RNAs are associated with P48. In vitro, both E. coli P48 and SRP54 specifically bind to 4.5S RNA. Strains depleted of 4.5S RNA strongly accumulate pre-beta-lactamase and fail to accumulate maltose binding protein. These effects commence well before any growth defect is observed and are suppressed by expression of human SRP7S. Strains overproducing P48 also accumulate pre-beta-lactamase. 4.5S RNA and P48 are components of a ribonucleoprotein particle that we propose to be required for the secretion of some proteins.

Signal peptide fragments of preprolactin and HIV‐1 p‐gp160 interact with calmodulin

Secretory proteins and most membrane proteins are synthesized with a signal sequence that is usually cleaved from the nascent polypeptide during transport into the lumen of the endoplasmic reticulum. Using site‐specific photo‐crosslinking we have followed the fate of the signal sequence of preprolactin in a cell‐free system. This signal sequence has an unusually long hydrophilic n‐region containing several positively charged amino acid residues. We found that after cleavage by signal peptidase the signal sequence is in contact with lipids and subunits of the signal peptidase complex. The cleaved signal sequence is processed further and an N‐terminal fragment is released into the cytosol. This signal peptide fragment was found to interact efficiently with calmodulin. Similar to preprolactin, the signal sequence of the HIV‐1 envelope protein p‐gp160 has the characteristic feature for calmodulin binding in its n‐region. We found that a signal peptide fragment of p‐gp160 was released into the cytosol and interacts with calmodulin. Our results suggest that signal peptide fragments of some cellular and viral proteins can interact with cytosolic target molecules. The functional consequences of such interactions remain to be established. However, our data suggest that signal sequences may be functionally more versatile than anticipated up to now.

The methionine-rich domain of the 54 kDa subunit of signal recognition particle is sufficient for the interaction with signal sequences.

The signal recognition particle (SRP) binds to signal sequences when they emerge from a translating ribosome and targets the complex of ribosome, nascent chain and SRP to the membrane of the rough endoplasmic reticulum (rER) allowing the co‐translational translocation of the nascent chain. By photo‐crosslinking it has been shown that the signal sequence of preprolactin (PPL) only interacts with the methionine‐rich (M) domain of the 54 kDa protein subunit (SRP54) of SRP. Here we show that (i) a signal‐anchor sequence is likewise crosslinked only to the methionine‐rich domain of SRP54, (ii) free SRP54 can interact with signal sequences independently of the other components of SRP, (iii) its M domain suffices to perform this function, and (iv) an essentially intact M domain is required for signal sequence recognition. Alkylation of the N+G domain in intact SRP54 with N‐ethyl maleimide (NEM), but not after cleavage with V8 protease, prevents the binding of a signal sequence to the M domain. This suggests a proximity between the N+G and M domains of SRP54 and raises the possibility that the role of the N+G domain may be to regulate the binding and/or the release of signal sequences.