The transport of integral membrane proteins across the nuclear pore complex

Meinema, Anne C; Poolman, Bert; Veenhoff, Liesbeth M.

doi:10.4161/nucl.20439

Cited by 14 publications

(13 citation statements)

References 82 publications

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“…The inner nuclear membrane faces the nucleoplasm and contains proteins that interact with the chromatin [45, 46] The outer nuclear membrane is continuous with the endoplasmic reticulum of the cell and has partially overlapping functions in the transport, synthesis and folding of proteins, and the synthesis of lipids [47]. The inner and the outer nuclear membrane of the nuclear envelope are penetrated by a large macromolecular structure nuclear pore complex (NPC) [48, 49]. …”

Section: The Nuclear Pore Complex and Nucleo-cytoplasmic Protein Tranmentioning

confidence: 99%

Nuclear transport of galectin-3 and its therapeutic implications

Funasaka¹,

Raz

Nangia‐Makker

2014

Seminars in Cancer Biology

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Galectin-3, a member of β-galactoside-binding gene family is a multi-functional protein, which regulates pleiotropic biological functions such as cell growth, cell adhesion, cell-cell interactions, apoptosis, angiogenesis and mRNA processing. Its unique structure enables it to interact with a plethora of ligands in a carbohydrate dependent or independent manner. Galectin-3 is mainly a cytosolic protein, but can easily traverse the intracellular and plasma membranes to translocate into the nucleus, mitochondria or get externalized. Depending on the cell type, specific experimental conditions in vitro, cancer type and stage, galectin-3 has been reported to be exclusively cytoplasmic, predominantly nuclear or distributed between the two compartments. In this review we have summarized the dynamics of galectin-3 shuttling between the nucleus and the cytoplasm, the nuclear transport mechanisms of galectin-3, how its specific interactions with the members of β-catenin signaling pathways affect tumor progression, and its implications as a therapeutic target.

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Section: The Nuclear Pore Complex and Nucleo-cytoplasmic Protein Tranmentioning

confidence: 99%

Nuclear transport of galectin-3 and its therapeutic implications

Funasaka¹,

Raz

Nangia‐Makker

2014

Seminars in Cancer Biology

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“…However, increasing evidence suggest that not only small soluble proteins but also integral membrane proteins with cytoplasmic domains up to 60 kDa can passively diffuse past the nuclear pore through a ∼10 nm side channel 6,24-28 . We propose that the Asi ubiquitin ligase targets such mislocalised and potentially harmful proteins for degradation.…”

mentioning

confidence: 99%

Protein quality control at the inner nuclear membrane

Khmelinskii

Blaszczak

Pantazopoulou

et al. 2014

Nature

189

244

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The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression1. The outer nuclear membrane is continuous with the endoplasmic reticulum (ER) and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by ER-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc72,3. However, little is known regarding protein quality control at the INM. Here we describe a protein degradation pathway at the INM mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi34. We report that the As complex functions together with the ubiquitin conjugating enzymes Ubc6andUbc7to degrade soluble and integral membrane proteins. Genetic evidence suggest that the Asi ubiquitin ligase defines a pathway distinct from but complementary to ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer (tFT)5, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquity ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalised integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.

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“…These methods have some effect in penetrating the cell membrane barrier, but have provided little benefit to cross the nuclear membrane barrier. Macromolecules, such as pDNA, must contain an NLS in order to be actively transported into the nucleus, by specific interaction of the NLS with the nuclear import proteins (9,10). …”

Section: Discussionmentioning

confidence: 99%

“…In addition, for successful gene delivery, the nuclear membrane is a second barrier in eukaryotic cells as all materials must go through nuclear pore complexes to enter the nucleus. Micromolecules with molecular weight <60 kDa and diameter <9 nm pass through nuclear pore complexes into the nucleus by passive diffusion, but macromolecule materials, such as plasmid DNA (pDNA), must contain nuclear localization signal (NLS) peptides, which mediate the active transport into the nucleus (9,10). …”

Section: Introductionmentioning

confidence: 99%

Enhanced effect of nuclear localization signal peptide during ultrasound-targeted microbubble destruction-mediated gene transfection

Cao

Zhou

Chen

et al. 2017

Molecular Medicine Reports

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Ultrasound-targeted microbubble destruction (UTMD) can promote the entry of plasmid DNA (pDNA) into the cell cytoplasm, by increasing the permeability of the cell membrane. But the transfection efficiency remains low due to inability of the pDNA to enter the nucleus. Various methods have been explored to improve the UTMD transfection efficiency, but with little success. In cells, the classic nuclear localization signal (cNLS) peptide is an amino acid sequence that signals proteins that are due for nuclear transport. The present study aimed to investigate whether binding of a cNLS peptide to the pDNA may improve the transfection efficiency of UTMD. Four experimental groups were analyzed: Control group (UTMD + pDNA), group with cNLS (UTMD + pDNA + cNLS), group with mutated NLS (mNLS; UTMD + pDNA + mNLS), and group with cNLS and the nuclear import blocker, wheat germ agglutinin (WGA; UTMD + pDNA + cNLS + WGA). The NLS was labeled by fluorescein isothiocyanate, whereas pDNA was labeled with Cy3. Different molar ratios were tested for the NLS and pDNA combination in order to achieve optimal binding of the two molecules. Human umbilical vein endothelial cells were then transfected using the optimum ultrasonic irradiation parameters and NLS/pDNA molar ratio. At 6 h post-transfection, the rates of Cy3-labeled pDNA inside the cells and their nuclei were detected by flow cytometry and laser confocal microscopy, and the cellular vs. nuclear uptake of pDNA was calculated. In order to further evaluate the effect of NLS on UTMD-mediated gene transfection, the transfection efficiency and relative expression levels of mRNA and protein were detected at 48 h post-transfection. The results demonstrated that the optimal molar ratio of NLS with pDNA was 104:1. The rates of pDNA successful entry into the cell and nucleus were significantly higher in the cNLS group compared with the control group. The transfection efficiency, and relative expression levels of mRNA and protein from the plasmid were significantly increased in the cNLS group compared with the control group. The mNLS group displayed no significant difference compared with the control group, while the WGA group exhibited significant inhibition in most indicators of transfection efficiency compared to the cNLS group. These results suggest that combining a cNLS peptide with pDNA during UTMD-mediated transfection significantly improved transfection efficiency. Thus, a cNLS peptide may be an important mediator and a new strategy in enhancing the efficiency of UTMD-mediated gene transfection.

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The transport of integral membrane proteins across the nuclear pore complex

Cited by 14 publications

References 82 publications

Nuclear transport of galectin-3 and its therapeutic implications

Nuclear transport of galectin-3 and its therapeutic implications

Protein quality control at the inner nuclear membrane

Enhanced effect of nuclear localization signal peptide during ultrasound-targeted microbubble destruction-mediated gene transfection

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