The SPCA1 Ca2+ Pump and Intracellular Membrane Trafficking

The Golgi apparatus (GA) is a dynamic store of Ca²⁺ that can be released into the cell cytosol. It can thus participate in the regulation of the Ca²⁺ concentration in the cytosol ([Ca²⁺]_cyt), which might be critical for intra‐Golgi transport. Secretory pathway Ca²⁺‐ATPase pump type 1 (SPCA1) is important in Golgi homeostasis of Ca²⁺. The subcellular localization of SPCA1 appears to be GA specific, although its precise location within the GA is not known. Here, we show that SPCA1 is mostly excluded from the cores of the Golgi cisternae and is instead located mainly on the lateral rims of Golgi stacks, in tubular noncompact zones that interconnect different Golgi stacks, and within tubular parts of the trans Golgi network, suggesting a role in regulation of the local [Ca²⁺]_cyt that is crucial for membrane fusion. SPCA1 knockdown by RNA interference induces GA fragmentation. These Golgi fragments lack the cis‐most and trans‐most cisternae and remain within the perinuclear region. This SPCA1 knockdown inhibits exit of vesicular stomatitis virus G‐protein from the GA and delays retrograde redistribution of the GA glycosylation enzymes into the endoplasmic reticulum caused by brefeldin A; however, exit of these enzymes from the endoplasmic reticulum is not affected. Thus, correct SPCA1 functioning is crucial to intra‐Golgi transport and maintenance of the Golgi ribbon.     

Ceramides modulate cell-surface acetylcholine receptor levels

The effects of ceramides (Cer) on the trafficking of the nicotinic acetylcholine receptor (AChR) to the plasma membrane were studied in CHO-K1/A5 cells, a clonal cell line that heterologously expresses the adult murine form of the receptor. When cells were incubated with short- (C₆-Cer) or long- (brain-Cer) chain Cer at low concentrations, an increase in the number of cell-surface AChRs was observed concomitant with a decrease in intracellular receptor levels. The alteration in AChR distribution by low Cer treatment does not appear to be a general mechanism since the surface expression of the green fluorescent protein derivative of the vesicular stomatitis virus protein (VSVG-GFP) was not affected. High Cer concentrations caused the opposite effects, decreasing the number of cell-surface AChRs, which exhibited higher affinity for [¹²⁵I]-α-bungarotoxin, and increasing the intracellular pool, which colocalized with trans-Golgi/TGN specific markers. The generation of endogenous Cer by sphingomyelinase treatment also decreased cell-surface AChR levels. These effects do not involve protein kinase Cζ or protein phosphatase 2A activation. Taken together, the results indicate that Cer modulate trafficking of AChRs to and stability at the cell surface.     

Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae.

Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.