Although cholesterol is synthesized in the endoplasmic reticulum (ER), compared with other cellular membranes, ER membrane has low cholesterol (3-6%). Most of the molecular machinery that regulates cellular cholesterol homeostasis also resides in the ER. Little is known about how cholesterol itself affects the ER membrane. Here, we demonstrate that acute cholesterol depletion in ER membranes impairs ER-to-Golgi transport of secretory membrane proteins. Cholesterol depletion is achieved by a brief inhibition of cholesterol synthesis with statins in cells grown in cholesterol-depleted medium. We provide evidence that secretory membrane proteins vesicular stomatitis virus glycoprotein and scavenger receptor A failed to be efficiently transported from the ER upon cholesterol depletion. Fluorescence photobleaching recovery experiments indicated that cholesterol depletion by statins leads to a severe loss of lateral mobility on the ER membrane of these transmembrane proteins, but not loss of mobility of proteins in the ER lumen. This impaired lateral mobility is correlated with impaired ER-to-Golgi transport. These results provide evidence for the first time that cholesterol is required in the ER membrane to maintain mobility of membrane proteins and thus protein secretion.

The prenylated Rab acceptor (PRA) 1 is a protein that binds prenylated Rab GTPases and inhibits their removal from the membrane by GDI. We describe here the isolation of a second isoform that can also bind Rab GTPases in a guanine nucleotide-independent manner. The two PRA isoforms showed distinct intracellular localization with PRA1 localized primarily to the Golgi complex and PRA2 to the endoplasmic reticulum (ER) compartment. The localization signal was mapped to the COOH-terminal domain of the two proteins. A DXEE motif served to target PRA1 to the Golgi. Mutation of any one of the acidic residues within this motif resulted in significant retention of PRA1 in the ER compartment. Moreover, the introduction of a di-acidic motif to the COOH-terminal domain of PRA2 resulted in partial localization to the Golgi complex. The domain responsible for ER localization of PRA2 was also confined to the carboxyl terminus. Our results showed that these sorting signals were primarily responsible for the differential localization of the two PRA isoforms.

Intestinal inflammation causes initial axonal degeneration and neuronal death but subsequent axon outgrowth from surviving neurons restores innervation density to the target smooth muscle cells. Elsewhere, the pro-inflammatory cytokines TNF␣ and IL-1␤ cause neurotoxicity, leading us to test their role in promoting enteric neuron death. In a rat coculture model, TNF␣ or IL-1␤ did not affect neuron number but did promote significant neurite outgrowth to twofold that of control by 48 h, while other cytokines (e.g., IL-4, TGF␤) were without effect. TNF␣ or IL-1␤ activated the NFB signaling pathway, and inhibition of NFB signaling blocked the stimulation of neurite growth. However, nuclear translocation of NFB in smooth muscle cells but not in adjacent neurons suggested a dominant role for smooth muscle cells. TNF␣ or IL-1␤ sharply increased both mRNA and protein for GDNF, while the neurotrophic effects of TNF␣ or IL-1␤ were blocked by the RET-receptor blocker vandetanib. Conditioned medium from cytokine-treated smooth muscle cells mimicked the neurotrophic effect, inferring that TNF␣ and IL-1␤ promote neurite growth through NFB-dependent induction of glial cell linederived neurotrophic factor (GDNF) expression in intestinal smooth muscle cells. In vivo, TNBS-colitis caused early nuclear translocation of NFB in smooth muscle cells. Conditioned medium from the intact smooth muscle of the inflamed colon caused a 2.5-fold increase in neurite number in cocultures, while Western blotting showed a substantial increase in GDNF protein. Pro-inflammatory cytokines promote neurite growth through upregulation of GDNF, a novel process that may facilitate re-innervation of smooth muscle cells and a return to homeostasis following initial damage.

The VAMP-associated proteins termed VAP are a small gene family of proteins characterised by the presence of an N-terminal major sperm protein (MSP) domain. The P56S mutation of the B isoform (VAPB) has been linked to late-onset amyotrophic lateral sclerosis (ALS8) and its expression causes formation of large ER aggregates. Overexpression of the wild-type A isoform (VAPA) but not the B isoform (VAPB), inhibited ER-to-Golgi transport of membrane proteins. This transport block by VAPA was primarily due to decreased segregation of membrane cargo into ER vesicles. We also found that VAPA inhibited lateral diffusion of membrane proteins, most likely through its stable association with microtubules. The MSP domain of VAP is known to interact with the FFAT motif (two phenylalanines in an acidic tract) of proteins involved in sterol regulation. Overexpression of FFAT restored ER-to-Golgi transport and lateral diffusion of membrane proteins, and resolved the large ER aggregates in VAPB-P56S. Application of a FFAT peptide restored in vitro ER vesicle budding and disrupted VAP-microtubule association. Thus, overexpression of the two VAP isoforms causes retention of ER membrane proteins by impeding lateral diffusion and their incorporation into transport vesicles. This inhibitory effect can be relieved by expression of the FFAT motif.

Prenylated Rab acceptor (PRA1) is a protein that binds Rab GTPases and the v-SNARE VAMP2. The protein is localized to the Golgi complex and post-Golgi vesicles. To determine its functional role, we generated a number of point mutations and divided them into three classes based on cellular localization. Class A mutants were retained in the endoplasmic reticulum (ER) and exerted an inhibitory effect on transport of vesicular stomatitis virus envelope glycoprotein (VSVG) from the ER to Golgi as well as to the plasma membrane. Class B mutants exhibited a highly condensed Golgi complex and inhibited exit of anterograde cargo from this organelle. Class C mutants exhibited an intermediate phenotype with Golgi and ER localization along with extensive tubular structures emanating from the Golgi complex. There was a direct correlation between the cellular phenotype and binding to Rab and VAMP2. Class A and C mutants showed a significant decrease in Rab and VAMP2 binding, whereas an increase in binding was observed in the class B mutants. Thus, PRA1 is required for vesicle formation from the Golgi complex and might be involved in recruitment of Rab effectors and SNARE proteins during cargo sequestration.Rab GTPases constitute the largest group within the Ras superfamily. They regulate vesicle trafficking by cycling through active membrane-bound GTP-and inactive cytosolic GDP-bound states. Membrane localization requires modification of the cysteine-containing motif at the carboxyl terminus by prenyl residues. Cycling between the membrane and cytosol is mediated by GDP dissociation inhibitor (GDI), 1 which extracts GDP-bound Rab from the membrane. Activation through guanine nucleotide exchange at the membrane is catalyzed by a guanine nucleotide exchange factor, of which a number have been identified in mammals (1, 2).Vesicular transport through the secretory pathway undergoes a number of discrete steps each involving budding, membrane remodeling, targeting, docking, and fusion. In ER to Golgi transport, anterograde cargo proteins such as VSVG are selectively transported to the Golgi along with resident ER proteins with the latter retrieved by a salvage process that recognizes distinct motifs within the protein (3). These transport vesicles contain an electron-dense coat assembled under the control of the small GTPase ARF (4). The fungal metabolite brefeldin A (BFA) inhibits ARF activation by stabilizing the inactive ARF-guanine nucleotide exchange factor (GEF) complex (5) resulting in retrograde transport of Golgi content to the ER. At the Golgi complex, cargo proteins destined for postGolgi locations are sorted into distinct carriers upon exit from the trans face, whereas Golgi resident proteins such as mannosidase II (Man II) are selectively retrieved in COPI-coated vesicles and returned to the cis face (6).Vesicle fusion is mediated by the core SNARE complex consisting of the vesicle protein VAMP, or synaptobrevin, and two other membrane proteins, syntaxin and SNAP-25 (7). Rab effectors play a regulatory role in this pro...