Martin Spiess

The sucrase-isomaltase complex: Primary structure, membrane-orientation, and evolution of a stalked, intrinsic brush border protein

The complete primary structure (1827 amino acids) of rabbit intestinal pro-sucrase-isomaltase (pro-SI) was deduced from the sequence of a nearly full-length cDNA. Pro-SI is anchored in the membrane by a single 20 amino acid segment spanning the bilayer only once. The amino-terminal, cytoplasmic domain consists of 12 amino acids and is not preceded by a cleaved leader sequence. This suggests a dual role for the membrane-spanning segment as an uncleaved signal for membrane insertion. This is followed by a 22 residue serine/threonine-rich, probably glycosylated, stretch, presumably forming the stalk on which the globular, catalytic domains are directed into the intestinal lumen. Following this is a high degree of homology between the isomaltase and sucrase portions (41% amino acid identity), indicating that pro-SI evolved by partial gene duplication.

In vitro binding of clathrin adaptors to sorting signals correlates with endocytosis and basolateral sorting.

To analyze the interaction of sorting signals with clathrin‐associated adaptor complexes, we developed an in vitro assay based on surface plasmon resonance analysis. This method monitors the binding of purified adaptors to immobilized oligopeptides in real time and determines binding kinetics and affinities. A peptide corresponding to the cytoplasmic domain of wild‐type influenza hemagglutinin, an apical membrane protein that is not endocytosed, did not significantly bind adaptor complexes. However, peptide sequences containing a tyrosine residue that has previously been shown to induce endocytosis and basolateral sorting were specifically recognized by adaptor complexes. The in vitro rates of adaptor association with these peptides correlated with the internalization rates of the corresponding hemagglutinin variants in vivo. Binding was observed both for purified AP‐2 adaptors of the plasma membrane and for AP‐1 adaptors of the Golgi, with similar apparent equilibrium dissociation constants in the range 10(‐7)‐10(‐6) M. Adaptor binding was also demonstrated for a sequence containing a C‐terminal di‐leucine sequence, the second major motif of endocytosis/basolateral sorting signals. These results confirm the concept that interaction of cytoplasmic signals with plasma membrane adaptors determines the endocytosis rate of membrane proteins, and suggest the model that clathrin‐coated vesicles of the trans‐Golgi network are involved in basolateral sorting.

Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence

To study the insertion of multispanning membrane proteins into the endoplasmic reticulum, we constructed novel proteins on the cDNA level by repeating, up to four times, the internal signal-anchor domain of the asialoglycoprotein receptor H1. Upon in vitro translation in the presence of microsomes, these polypeptides are indeed inserted as polytopic membrane proteins. The first hydrophobic domain functions as a signal and the second as a stop-transfer sequence, while the third initiates a second translocation process, halted again by the fourth. We were able to demonstrate that insertion occurs sequentially, starting with the first apolar segment from the amino terminus. By replacing the original signal-anchor domains by a mutant sequence not recognized by signal recognition particle (SRP), it was shown that only the first hydrophobic domain needs to be a signal sequence and that the second translocation event does not require SRP.

Topogenesis of membrane proteins: determinants and dynamics

For targeting and integration of proteins into the mammalian endoplasmic reticulum, two types of signals can be distinguished: those that translocate their C‐terminal sequence (cleavable signals and signal‐anchors) and those that translocate their N‐terminus (reverse signal‐anchors). In addition to the well established effect of flanking charges, also the length and hydrophobicity of the apolar core of the signal as well as protein folding and glycosylation contribute to orienting the signal in the translocon. In multi‐spanning membrane proteins, topogenic determinants are distributed throughout the sequence and may even compete with each other. During topogenesis, segments of up to 60 residues may move back and forth through the translocon, emphasizing unexpected dynamic aspects of topogenesis.

The asialoglycoprotein receptor is a potential liver-specific receptor for Marburg virus

The liver is one of the main target organs of Marburg virus (MBG), a filovirus causing severe haemorrhagic fever with a high fatality rate in humans and non-human primates. MBG grown in certain cells does not contain neuraminic acid, but has terminal galactose on its surface glycoprotein. This observation indicated that the asialoglycoprotein receptor (ASGP-R) of hepatocytes may serve as a receptor for MBG in the liver. Binding studies revealed that the attachment of MBG to ASGP-R-expressing HepG2 cells, but not to ASGP-R-negative E6 Vero cells, has the characteristics of ligand binding to the ASGP-R: binding is dependent on calcium and is inhibited by excess asialofetuin and by anti-ASGP-R antiserum. Asialofetuin and the specific antiserum also inhibited MBG infection of HepG2 cells. In addition, it was shown that expression of ASGP-R cDNA in NIH 3T3 cells enhanced the susceptibility of these cells to MBG infection 4.5-fold. Interaction of MBG with the hepatic ASGP-R could thus explain the marked hepatotropism of the virus.

Recognition of sorting signals by clathrin adaptors

Sorting of membrane proteins is generally mediated by cytosolic coats, which create a scaffold to form coated buds and vesicles and to selectively concentrate cargo by interacting with cytosolic signals. The classical paradigm is the interaction between clathrin coats and associated adaptor proteins, which cluster receptors with characteristic tyrosine and dileucine motifs during endocytosis. Clathrin in association with different sets of adaptors is found in addition at the trans‐Golgi network and endosomes. Sequences similar to internalization signals also direct lysosomal and basolateral sorting, which implicates related clathrin‐adaptor coats in the respective sorting pathways. This review concentrates on the recognition of sorting signals by clathrin‐associated adaptor proteins, an area of significant recent progress due to new methodological and conceptual approaches.  BioEssays 21:558–567, 1999.

Heads or tails — what determines the orientation of proteins in the membrane

The same translocation machinery in the endoplasmic reticulum translocates either the N‐ or the C‐terminal domain of signal‐anchor proteins across the membranes. Charged residues flanking the signal sequence are important to determine which end is translocated, but are not sufficient to generate a uniform topology. The folding state of the N‐terminal segment, which is to be translocated posttranslationally, and the length or hydrophobicity of the signal sequence are additional criteria to determine protein orientation in the membrane.

An internal signal sequence: The asialoglycoprotein receptor membrane anchor

The human asialoglycoprotein receptor H1 is anchored in the membrane by a single stretch of 20 hydrophobic amino acids; the hydrophilic amino terminus faces the cytoplasm, and the carboxyl terminus is exoplasmic. We show here that glycosylation and insertion of the asialoglycoprotein receptor into the endoplasmic reticulum membrane is cotranslational and SRP-dependent and occurs without proteolytic cleavage. The membrane-anchor domain is necessary for membrane insertion, since a receptor with the segment deleted is neither inserted nor glycosylated. The segment is also sufficient for membrane insertion, since it will initiate translocation of a carboxy-terminal domain of rat alpha-tubulin across the membrane. We propose that a helical hairpin mechanism of membrane insertion is used both by cleaved amino-terminal and uncleaved internal signal sequences.

Molecular mechanism of signal sequence orientation in the endoplasmic reticulum

We have analyzed in vivo how model signal sequences are inserted and oriented in the membrane during cotranslational integration into the endoplasmic reticulum. The results are incompatible with the current models of retention of positive flanking charges or loop insertion of the polypeptide into the translocon. Instead they indicate that these N‐terminal signals initially insert head‐on with a cytoplasmic C‐terminus before they invert their orientation to translocate the C‐terminus. The rate of inversion increases with more positive N‐terminal charge and is reduced with increasing hydrophobicity of the signal. Inversion may proceed for up to ∼50 s, when it is terminated by a signal‐independent process. These findings provide a mechanism for the topogenic effects of flanking charges as well as of signal hydrophobicity.

TRANSMEMBRANE ORIENTATION OF SIGNAL-ANCHOR PROTEINS IS AFFECTED BY THE FOLDING STATE BUT NOT THE SIZE OF THE N-TERMINAL DOMAIN

Upon insertion of a signal‐anchor protein into the endoplasmic reticulum membrane, either the C‐terminal or the N‐terminal domain is translocated across the membrane. Charged residues flanking the transmembrane domain are important determinants for this decision, but are not necessarily sufficient to generate a unique topology. Using a model protein that is inserted into the membrane to an equal extent in either orientation, we have tested the influence of the size and the folding state of the N‐terminal domain on the insertion process. A small zinc finger domain or the full coding sequence of dihydrofolate reductase were fused to the N‐terminus. These stably folding domains hindered or even prevented their translocation. Disruption of their structure by destabilizing mutations largely restored transport across the membrane. Translocation efficiency, however, did not depend on the size of the N‐terminal domain within a range of 40–237 amino acids. The folding behavior of the N‐terminal domain is thus an important factor in the topogenesis of signal‐anchor proteins.