RESUMO
Sorting nexins anchor trafficking machines to membranes by binding phospholipids. The paradigm of the superfamily is sorting nexin 3 (SNX3), which localizes to early endosomes by recognizing phosphatidylinositol 3-phosphate (PI3P) to initiate retromer-mediated segregation of cargoes to the trans-Golgi network (TGN). Here we report the solution structure of full length human SNX3, and show that PI3P recognition is accompanied by bilayer insertion of a proximal loop in its extended Phox homology (PX) domain. Phosphoinositide (PIP) binding is completely blocked by cancer-linked phosphorylation of a conserved serine beside the stereospecific PI3P pocket. This "PIP-stop" releases endosomal SNX3 to the cytosol, and reveals how protein kinases control membrane assemblies. It constitutes a widespread regulatory element found across the PX superfamily and throughout evolution including of fungi and plants. This illuminates the mechanism of a biological switch whereby structured PIP sites are phosphorylated to liberate protein machines from organelle surfaces.
Assuntos
Membrana Celular/metabolismo , Fosfatidilinositóis/metabolismo , Nexinas de Classificação/metabolismo , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipídeos/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico , Nexinas de Classificação/química , Rede trans-Golgi/metabolismoRESUMO
In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.
Assuntos
Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Nexinas de Classificação/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Endocitose , Endossomos/ultraestrutura , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Transporte Proteico , Interferência de RNA , Homologia de Sequência de Aminoácidos , Vesiculovirus/fisiologiaRESUMO
Recent evidence suggests that non-muscle myosin IIB (NMIIB) contributes to smooth muscle contraction. This study was designed to determine the effects of NMIIB on the cross-bridge cycling rate. The cross-bridge cycling rate was investigated using sinusoidal analysis. Frequency analysis revealed two asymptotes in the Bode plot of the data; and the intersection of the asymptotes (corner frequency) was higher for the B(+/-) strain (8.73+/-1.10 Hz vs 16.56+/-1.26 Hz, P<0.05), consistent with a higher overall cross-bridge cycling rate in heterozygous NMIIB KO (B(+/-)) vs WT mice. These results demonstrate that because of their long duty cycle, NMIIB cross-bridges act as an internal load on smooth muscle myosin to decrease the overall cross-bridge cycling rate and muscle V(max) during force maintenance.