ARL4A acts with GCC185 to modulate Golgi complex organization.

ADP-ribosylation factor-like protein 4A (ARL4A) is a developmentally regulated member of the ARF/ARL GTPase family. The primary structure of ARL4A is very similar to that of other ARF/ARL molecules, but its function remains unclear. The trans-Golgi network golgin GCC185 is required for maintenance of Golgi structure and distinct endosome-to-Golgi transport. We show here that GCC185 acts as a new effector for ARL4 to modulate Golgi organization. ARL4A directly interacts with GCC185 in a GTP-dependent manner. Sub-coiled-coil regions of the CC2 domain of GCC185 are required for the interaction between GCC185 and ARL4A. Depletion of ARL4A reproduces the GCC185-depleted phenotype, causing fragmentation of the Golgi compartment and defects in endosome-to-Golgi transport. GCC185 and ARL4A localize to the Golgi independently of each other. Deletion of the ARL4A-interacting region of GCC185 results in inability to maintain Golgi structure. Depletion of ARL4A impairs the interaction between GCC185 and cytoplasmic linker-associated proteins 1 and 2 (CLASP1 and CLASP2, hereafter CLASPs) in vivo, and abolishes the GCC185-mediated Golgi recruitment of these CLASPs, which is crucial for the maintenance of Golgi structure. In summary, we suggest that ARL4A alters the integrity of the Golgi structure by facilitating the interaction of GCC185 with CLASPs.

The fusion peptide domain is the primary membrane-inserted region and enhances membrane interaction of the ectodomain of HIV-1 gp41.

To execute the membrane fusion function, it is necessary for the fusion protein of the virus to penetrate into the hydrophobic milieu of membrane bilayer. Hence identification of the region(s) of the ectodomain of viral fusion proteins involved in the membrane insertion and their interaction with the rest of the fusion protein in the membrane would be important for the mechanistic study of membrane fusion. To this end, we examined membrane activity of the fusion peptide, and the ectodomain protein with or without the fusion peptide domain of HIV-1 gp41 by several biophysical measurements. The results revealed that the ectodomain protein containing the fusion peptide domain had higher membrane-perturbing activity and deeper membrane insertion, while the construct lacking the fusion peptide domain had much lower membrane activity. Strikingly, the N-terminal heptad repeat region was found to be induced deeper into the membrane by the fusion peptide, consistent with the role of the latter in the membrane penetration. We concluded that the fusion peptide is the only stretch of gp41 ectodomain that embeds deeply in the membrane interior in the prefusion stage. The function of fusion peptide in terms of membrane interaction and the implications of its interplay with other domains of gp41 on the membrane fusion cascade were discussed.

Membrane interaction and structure of the transmembrane domain of influenza hemagglutinin and its fusion peptide complex.

BACKGROUND: To study the organization and interaction with the fusion domain (or fusion peptide, FP) of the transmembrane domain (TMD) of influenza virus envelope glycoprotein for its role in membrane fusion which is also essential in the cellular trafficking of biomolecules and sperm-egg fusion. RESULTS: The fluorescence and gel electrophoresis experiments revealed a tight self-assembly of TMD in the model membrane. A weak but non-random interaction between TMD and FP in the membrane was found. In the complex, the central TMD oligomer was packed by FP in an antiparallel fashion. FP insertion into the membrane was altered by binding to TMD. An infrared study exhibited an enhanced membrane perturbation by the complex formation. A model was built to illustrate the role of TMD in the late stages of influenza virus-mediated membrane fusion reaction. CONCLUSION: The TMD oligomer anchors the fusion protein in the membrane with minimal destabilization to the membrane. Upon associating with FP, the complex exerts a synergistic effect on the membrane perturbation. This effect is likely to contribute to the complete membrane fusion during the late phase of fusion protein-induced fusion cascade. The results presented in the work characterize the nature of the interaction of TMD with the membrane and TMD in a complex with FP in the steps leading to pore initiation and dilation during virus-induced fusion. Our data and proposed fusion model highlight the key role of TMD-FP interaction and have implications on the fusion reaction mediated by other type I viral fusion proteins. Understanding the molecular mechanism of membrane fusion may assist in the design of anti-viral drugs.

Molecular Dynamics Simulations of 441 Two-Residue Peptides in Aqueous Solution: Conformational Preferences and Neighboring Residue Effects with the Amber ff99SB-ildn-NMR Force Field.

Understanding the intrinsic conformational preferences of amino acids and the extent to which they are modulated by neighboring residues is a key issue for developing predictive models of protein folding and stability. Here we present the results of 441 independent explicit-solvent MD simulations of all possible two-residue peptides that contain the 20 standard amino acids with histidine modeled in both its neutral and protonated states. (3)J(HNHα) coupling constants and δ(Hα) chemical shifts calculated from the MD simulations correlate quite well with recently published experimental measurements for a corresponding set of two-residue peptides. Neighboring residue effects (NREs) on the average (3)J(HNHα) and δ(Hα) values of adjacent residues are also reasonably well reproduced, with the large NREs exerted experimentally by aromatic residues, in particular, being accurately captured. NREs on the secondary structure preferences of adjacent amino acids have been computed and compared with corresponding effects observed in a coil library and the average ß-turn preferences of all amino acid types have been determined. Finally, the intrinsic conformational preferences of histidine, and its NREs on the conformational preferences of adjacent residues, are both shown to be strongly affected by the protonation state of the imidazole ring.

GTP-binding-defective ARL4D alters mitochondrial morphology and membrane potential.

ARL4D, ARL4A, and ARL4C are closely related members of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of GTPases. All three ARL4 proteins contain nuclear localization signals (NLSs) at their C-termini and are primarily found at the plasma membrane, but they are also present in the nucleus and cytoplasm. ARF function and localization depends on their controlled binding and hydrolysis of GTP. Here we show that GTP-binding-defective ARL4D is targeted to the mitochondria, where it affects mitochondrial morphology and function. We found that a portion of endogenous ARL4D and the GTP-binding-defective ARL4D mutant ARL4D(T35N) reside in the mitochondria. The N-terminal myristoylation of ARL4D(T35N) was required for its localization to mitochondria. The localization of ARL4D(T35N) to the mitochondria reduced the mitochondrial membrane potential (ΔΨm) and caused mitochondrial fragmentation. Furthermore, the C-terminal NLS region of ARL4D(T35N) was required for its effect on the mitochondria. This study is the first to demonstrate that the dysfunctional GTP-binding-defective ARL4D is targeted to mitochondria, where it subsequently alters mitochondrial morphology and membrane potential.