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Recent evidence suggests a regulatory connection between cell volume, endoplasmic reticulum (ER) export, and stimulated Golgi-to-ER transport. To investigate the potential role of protein kinases we tested a panel of protein kinase inhibitors for their effect on these steps. One inhibitor, H89, an isoquinolinesulfonamide that is commonly used as a selective(More)
By forming a molecular tether between two membranes, p115, giantin, and GM130 may mediate multiple Golgi-related processes including vesicle transport, cisternae formation, and cisternal stacking. The tether is proposed to involve the simultaneous binding of p115 to giantin on one membrane and to GM130 on another membrane. To explore this model, we tested(More)
The cytosolic coat protein complex II (COPII) mediates vesicle formation from the endoplasmic reticulum (ER) and is essential for ER-to-Golgi trafficking. The minimal machinery for COPII assembly is well established. However, additional factors may regulate the process in mammalian cells. Here, a morphological COPII assembly assay using purified COPII(More)
Despite the potential importance of retrieval-based targeting, few Golgi cisternae-localized proteins have been demonstrated to be targeted by retrieval, and the putative retrieval signals remain unknown. Golgi phosphoprotein of 130 kDa (GPP130) is a cis-Golgi protein that allows assay of retrieval-based targeting because it redistributes to endosomes upon(More)
The structure of the endoplasmic reticulum (ER) undergoes highly regulated changes in specialized cell types. One frequently observed type of change is its reorganization into stacked and concentrically whorled membranes, but the underlying mechanisms and functional relevance for cargo export are unknown. Here, we identify Yip1A, a conserved membrane(More)
Physiological conditions that impinge on constitutive traffic and affect organelle structure are not known. We report that osmotically induced cell volume changes, which are known to occur under a variety of conditions, rapidly inhibited endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells. Both ER export and ER Golgi intermediate compartment(More)
The mechanisms that structure the mammalian endoplasmic reticulum (ER) network are not fully understood. Here we show that salt extraction of semi-intact normal rat kidney (NRK) fibroblasts and subsequent incubation of the extracted cells with ATP resulted in dramatic ER network retraction. Under these conditions, addition of a single protein, Nucleoside(More)
The membrane-anchored atlastin GTPase couples nucleotide hydrolysis to the catalysis of homotypic membrane fusion to form a branched endoplasmic reticulum network. Trans dimerization between atlastins anchored in opposing membranes, accompanied by a cross-over conformational change, is thought to draw the membranes together for fusion. Previous studies on(More)
The endoplasmic reticulum (ER) of specialized cells can undergo dramatic changes in structural organization, including formation of concentric whorls. We previously reported that depletion of Yip1A, an integral membrane protein conserved between yeast and mammals, caused ER whorl formation reminiscent of that seen in specialized cells. Yip1A and its yeast(More)
Endoplasmic reticulum (ER) network branching requires homotypic tethering and fusion of tubules mediated by the atlastin (ATL) guanosine triphosphatase (GTPase). Recent structural studies on the ATL soluble domain reveal two dimeric conformers proposed to correspond to a tethered prefusion state and a postfusion state. How the prefusion conformer(More)