Luis Martínez-Gil

Plant Virus Cell-to-Cell Movement Is Not Dependent on the Transmembrane Disposition of Its Movement Protein

ABSTRACT The cell-to-cell transport of plant viruses depends on one or more virus-encoded movement proteins (MPs). Some MPs are integral membrane proteins that interact with the membrane of the endoplasmic reticulum, but a detailed understanding of the interaction between MPs and biological membranes has been lacking. The cell-to-cell movement of the Prunus necrotic ringspot virus (PNRSV) is facilitated by a single MP of the 30K superfamily. Here, using a myriad of biochemical and biophysical approaches, we show that the PNRSV MP contains only one hydrophobic region (HR) that interacts with the membrane interface, as opposed to being a transmembrane protein. We also show that a proline residue located in the middle of the HR constrains the structural conformation of this region at the membrane interface, and its replacement precludes virus movement.

The Tobacco mosaic virus Movement Protein Associates with but Does Not Integrate into Biological Membranes

ABSTRACT Plant positive-strand RNA viruses require association with plant cell endomembranes for viral translation and replication, as well as for intra- and intercellular movement of the viral progeny. The membrane association and RNA binding of the Tobacco mosaic virus (TMV) movement protein (MP) are vital for orchestrating the macromolecular network required for virus movement. A previously proposed topological model suggests that TMV MP is an integral membrane protein with two putative α-helical transmembrane (TM) segments. Here we tested this model using an experimental system that measured the efficiency with which natural polypeptide segments were inserted into the ER membrane under conditions approximating the in vivo situation, as well as in planta. Our results demonstrated that the two hydrophobic regions (HRs) of TMV MP do not span biological membranes. We further found that mutations to alter the hydrophobicity of the first HR modified membrane association and precluded virus movement. We propose a topological model in which the TMV MP HRs intimately associate with the cellular membranes, allowing maximum exposure of the hydrophilic domains of the MP to the cytoplasmic cellular components. IMPORTANCE To facilitate plant viral infection and spread, viruses encode one or more movement proteins (MPs) that interact with ER membranes. The present work investigated the membrane association of the 30K MP of Tobacco mosaic virus (TMV), and the results challenge the previous topological model, which predicted that the TMV MP behaves as an integral membrane protein. The current data provide greatly needed clarification of the topological model and provide substantial evidence that TMV MP is membrane associated only at the cytoplasmic face of the membrane and that neither of its domains is integrated into the membrane or translocated into the lumen. Understanding the topology of MPs in the ER is vital for understanding the role of the ER in plant virus transport and for predicting interactions with host factors that mediate resistance to plant viruses.

The Surfactant Peptide KL4 Sequence Is Inserted with a Transmembrane Orientation into the Endoplasmic Reticulum Membrane

Surfactant protein B (SP-B) is an essential component of pulmonary surfactant. Synthetic surfactant peptide KL(4), a peptide based on a C-terminal amphipathic helical region of human SP-B, efficiently mimics some functional properties of SP-B and is included in therapeutic surfactant preparations used in trials to treat respiratory distress syndrome. The membrane orientation of this peptide is controversial. We used an in vitro transcription-translation system to study the insertion of hydrophobic sequences into microsomal membranes, and showed that the KL(4) sequence integrates efficiently with a transmembrane orientation despite the presence of intermittent lysines throughout the sequence. In contrast, the precise sequence of the C-terminal SP-B amphipathic region failed to integrate, indicating a nontransmembrane orientation. Differences in the membrane insertion between KL(4) and the SP-B-inspiring sequence match predictions from calculated free energies of insertion of the two sequences into membranes.

Membrane Insertion and Biogenesis of the Turnip Crinkle Virus p9 Movement Protein

ABSTRACT Plant viral infection and spread depends on the successful introduction of a virus into a cell of a compatible host, followed by replication and cell-to-cell transport. The movement proteins (MPs) p8 and p9 of Turnip crinkle virus are required for cell-to-cell movement of the virus. We have examined the membrane association of p9 and found that it is an integral membrane protein with a defined topology in the endoplasmic reticulum (ER) membrane. Furthermore, we have used a site-specific photo-cross-linking strategy to study the membrane integration of the protein at the initial stages of its biosynthetic process. This process is cotranslational and proceeds through the signal recognition particle and the translocon complex.

Membrane integration of poliovirus 2B viroporin.

ABSTRACT Virus infections can result in a variety of cellular injuries, and these often involve the permeabilization of host membranes by viral proteins of the viroporin family. Prototypical viroporin 2B is responsible for the alterations in host cell membrane permeability that take place in enterovirus-infected cells. 2B protein can be localized at the endoplasmic reticulum (ER) and the Golgi complex, inducing membrane remodeling and the blockade of glycoprotein trafficking. These findings suggest that 2B has the potential to integrate into the ER membrane, but specific information regarding its biogenesis and mechanism of membrane insertion is lacking. Here, we report experimental results of in vitro translation-glycosylation compatible with the translocon-mediated insertion of the 2B product into the ER membrane as a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. A similar topology was found when 2B was synthesized in cultured cells. In addition, the in vitro translation of several truncated versions of the 2B protein suggests that the two hydrophobic regions cooperate to insert into the ER-derived microsomal membranes.

Membrane Insertion and Topology of the Translocating Chain-Associating Membrane Protein (TRAM)

The translocating chain-associating membrane protein (TRAM) is a glycoprotein involved in the translocation of secreted proteins into the endoplasmic reticulum (ER) lumen and in the insertion of integral membrane proteins into the lipid bilayer. As a major step toward elucidating the structure of the functional ER translocation/insertion machinery, we have characterized the membrane integration mechanism and the transmembrane topology of TRAM using two approaches: photocross-linking and truncated C-terminal reporter tag fusions. Our data indicate that TRAM is recognized by the signal recognition particle and translocon components, and suggest a membrane topology with eight transmembrane segments, including several poorly hydrophobic segments. Furthermore, we studied the membrane insertion capacity of these poorly hydrophobic segments into the ER membrane by themselves. Finally, we confirmed the main features of the proposed membrane topology in mammalian cells expressing full-length TRAM.

Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion

α-Helical hairpins, consisting of a pair of closely spaced transmembrane (TM) helices that are connected by a short interfacial turn, are the simplest structural motifs found in multi-spanning membrane proteins. In naturally occurring hairpins, the presence of polar residues is common and predicted to complicate membrane insertion. We postulate that the pre-packing process offsets any energetic cost of allocating polar and charged residues within the hydrophobic environment of biological membranes. Consistent with this idea, we provide here experimental evidence demonstrating that helical hairpin insertion into biological membranes can be driven by electrostatic interactions between closely separated, poorly hydrophobic sequences. Additionally, we observe that the integral hairpin can be stabilized by a short loop heavily populated by turn-promoting residues. We conclude that the combined effect of TM-TM electrostatic interactions and tight turns plays an important role in generating the functional architecture of membrane proteins and propose that helical hairpin motifs can be acquired within the context of the Sec61 translocon at the early stages of membrane protein biosynthesis. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains from primary structures.

Viral membrane protein topology is dictated by multiple determinants in its sequence.

The targeting, insertion, and topology of membrane proteins have been extensively studied in both prokaryotes and eukaryotes. However, the mechanisms used by viral membrane proteins to generate the correct topology within cellular membranes are less well understood. Here, the effect of flanking charges and the hydrophobicity of the N-terminal hydrophobic segment on viral membrane protein topogenesis are examined systematically. Experimental data reveal that the classical topological determinants have only a minor effect on the overall topology of p9, a plant viral movement protein. Since only a few individual sequence alterations cause an inversion of p9 topology, its topological stability is robust. This result further indicates that the protein has multiple, and perhaps redundant, structural features that ensure that it always adopts the same topology. These critical topogenic sequences appear to be recognized and acted upon from the initial stages of protein biosynthesis, even before the ribosome ends protein translation.

Membrane topology of gp41 and amyloid precursor protein: Interfering transmembrane interactions as potential targets for HIV and Alzheimer treatment

Abstract The amyloid precursor protein (APP), that plays a critical role in the development of senile plaques in Alzheimer disease (AD), and the gp41 envelope protein of the human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS), are single-spanning type-1 transmembrane (TM) glycoproteins with the ability to form homo-oligomers. In this review we describe similarities, both in structural terms and sequence determinants of their TM and juxtamembrane regions. The TM domains are essential not only for anchoring the proteins in membranes but also have functional roles. Both TM segments contain GxxxG motifs that drive TM associations within the lipid bilayer. They also each possess similar sequence motifs, positioned at the membrane interface preceding their TM domains. These domains are known as cholesterol recognition/interaction amino acid consensus (CRAC) motif in gp41 and CRAC-like motif in APP. Moreover, in the cytoplasmic domain of both proteins other α-helical membranotropic regions with functional implications have been identified. Recent drug developments targeting both diseases are reviewed and the potential use of TM interaction modulators as therapeutic targets is discussed.

Viroporins, Examples of the Two-Stage Membrane Protein Folding Model.

Viroporins are small, α-helical, hydrophobic virus encoded proteins, engineered to form homo-oligomeric hydrophilic pores in the host membrane. Viroporins participate in multiple steps of the viral life cycle, from entry to budding. As any other membrane protein, viroporins have to find the way to bury their hydrophobic regions into the lipid bilayer. Once within the membrane, the hydrophobic helices of viroporins interact with each other to form higher ordered structures required to correctly perform their porating activities. This two-step process resembles the two-stage model proposed for membrane protein folding by Engelman and Poppot. In this review we use the membrane protein folding model as a leading thread to analyze the mechanism and forces behind the membrane insertion and folding of viroporins. We start by describing the transmembrane segment architecture of viroporins, including the number and sequence characteristics of their membrane-spanning domains. Next, we connect the differences found among viroporin families to their viral genome organization, and finalize focusing on the pathways used by viroporins in their way to the membrane and on the transmembrane helix-helix interactions required to achieve proper folding and assembly.