Characterization of the Golgi Complex Cleared of Proteins in Transit and Examination of Calcium Uptake Activities

Abstract

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were Ͼ99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that Ͼ90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200-to 400-fold with and 400-to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.

The Golgi PMR1 P-type ATPase of Caenorhabditis elegans

“…In contrast to the growing unanimity on the role of Golgi as a Ca 2ϩ store, there remains some confusion as to the type of Ca 2ϩ -accumulation system responsible to replenish the store. Ca 2ϩ pumps belonging to the class of P-type ion-motive ATPases have been proposed to take up this task: SERCA2 (6,10), the plasma-membrane Ca 2ϩ pump PMCA en route to the plasma membrane (12) and PMR1 (6,13). The SERCA pumps, which are encoded by three different genes in vertebrates (human gene names: ATP2A1-3), but apparently only by a single gene in invertebrates like C. elegans, are the best characterized members of this class and are responsible for the accumulation of Ca 2ϩ into the ER or into the sarcoplasmic reticulum.…”

Proteomic Analysis of Two Functional States of the Golgi Complex in Mammary Epithelial Cells

“…Release of acini from the tissue was monitored by light microscopy and an enriched acinar preparation was collected by low-speed centrifugation. Subsequently, Golgi fractions were isolated from this enriched epithelial preparation (30).…”

“…Extensive assays of enrichment and yield of various markers are routinely used to characterize each step in a fractionation protocol (30). Each isolated Golgi fraction (GF) was characterized biochemically by quantitative immunoblot, and morphologically by electron microscopy (EM).…”

“…It was administered as published previously (30). The tissue is minced and incubated at 37°C in 0.075% collagenase/0.15% hyaluronidase/ HBBS containing 1 mg/ml each of proteolytic inhibitors chymostatin, leupeptin, antipain, and pepstatin.…”

The Current State of the Golgi Proteomes

“…While only a handful of proteins were identified by cross comparing reference maps and immunoblotting the study demonstrated the validity of a proteomic approach to analyze the Golgi and could discern resident proteins from cargo and cytosolic proteins (Taylor et al, 1997b). Significantly, the study employed a recently developed sequential sucrose gradient enrichment method (Taylor et al, 1997a) and enabled the reliable visualization of Golgi proteins by 2-DE with reduced contaminants. The sequential sucrose enrichment of stacked Golgi from liver samples involved initially loading a clarified homogenate (post-nuclear supernatant) between 0.86 M and 0.25 M sucrose steps followed by centrifugation.…”