Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Cell Biology of the Endoplasmic Reticulum and the Golgi Apparatus through Proteomics Jeffrey Smirle1, Catherine E. Au1, Michael Jain2, Kurt Dejgaard1, Tommy Nilsson1, and John Bergeron1 1The Research Institute of the McGill University Health Centre and the Department of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada 2University of Toronto, Division of Hematology, Toronto, Ontario M5G 2M9, Canada Correspondence: [email protected] Enriched endoplasmic reticulum (ER) and Golgi membranes subjected to mass spectrometry have uncovered over a thousand different proteins assigned to the ER and Golgi apparatus of rat liver. This, in turn, led to the uncovering of several hundred proteins of poorly understood function and, through hierarchical clustering, showed that proteins distributed in patterns suggestive of microdomains in cognate organelles. This has led to new insights with respect to their intracellular localization and function. Another outcome has been the critical testing of the cisternal maturation hypothesis showing overwhelming support for a predominant role of COPI vesicles in the transport of resident proteins of the ER and Golgi apparatus (as opposed to biosynthetic cargo). Here we will discuss new insights gained and also highlight new avenues undertaken to further explore the cell biology of the ER and the Golgi apparatus through tandem mass spectrometry. ost biosynthetic proteins destined for the membranes, and ends with tripartite-like struc- Mplasma membrane and secretion, as well tures of tubular smooth (ribosome-free) ER. as resident proteins of endosomes, lysosomes, The ratio of rough and smooth ER is cell the endoplasmic reticulum (ER), and the Golgi type specific. Stem cells have little rough ER apparatus originate with cotranslational trans- and smooth ER (Murphy et al. 1971; Cheng location into the ER. This is followed by care- and Leblond 1974), whereas cells highly special- fully controlled folding and quality control. The ized in protein secretion, such as in the exocrine ER is also a major site for sensing cellular stress pancreas, have extensive rough ER with little and for cholesterol and phospholipid biosyn- to no smooth ER (Palade and Siekevitz 1956; thesis and constitutes a vast continuous endo- Jamieson and Palade 1967). By contrast, liver pa- membrane system that often pervades the entire renchymal hepatocytes have a near equal abun- cytoplasm. The ER commences as the nuclear dance of rough and smooth ER with the latter envelope extends to rough (ribosome-studded) associated with glycogen storage and elimination Editors: Susan Ferro-Novick, Tom A. Rapoport, and Randy Schekman Additional Perspectives on The Endoplasmic Reticulum available at www.cshperspectives.org Copyright # 2013 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a015073 Cite this article as Cold Spring Harb Perspect Biol 2013;5:a015073 1 Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press J. Smirle et al. of exogenous steroids through a multitude of or to the endosomal endomembrane system. P450-driven oxidation pathways (Bruni and Por- Some specialized cargo is also packaged and ter1965;Loud1968;Estabrooketal.1971;Blouin concentrated into dedicated membrane struc- et al. 1977; Reed and Backes 2012). At the other tures for regulated secretion. extreme,Leydigcellsofthetestisarespecializedin The functional and morphological demar- cholesterol metabolism to form testosterone and cation between the ER and the Golgi apparatus are enriched with smooth ER (Mori and Chris- is usually assumed to be complete, giving rise to tensen 1980). Emanating from the ER of all cell the notion of two independent organelles— types in mammals are discrete export sites that each with their own distinct functions. Because are distributed throughout the cell, sometimes of the extensive recycling that takes place, how- numbering in the hundreds. ER export is con- ever, the two organelles appear functionally trolled by the COPII coat machinery, which has intertwined. Indeed, inhibition of the Golgi- been characterized through proteomics analy- located ARF1 guanine nucleotide exchange fac- ses in yeast (Otte et al. 2001) and is coupled to tor GBF1 results in a rapid collapse of the Golgi microtubule- and dynein/dynactin-dependent apparatus into the ER (Misumi et al. 1986; Oda transport directed toward the central juxta-nu- et al. 1987; Fujiwara et al. 1988; Lippincott- clear Golgi apparatus (COPII vesicles are dis- Schwartz et al. 1989; Claude et al. 1999). As cussed in Lord et al. [2013]). This occurs in ve- ARF1GDP to ARF1GTP conversion is obligatory sicular/tubular clusters (VTCs) that seemingly to COPI recruitment and vesicle formation and undergo a maturation/distillation process (Sa- subsequent recycling, the effect of BFA seems at raste and Kuismanen 1984) such that when ar- first counter intuitive, but can be explained riving at the cis-face Golgi stacks, biosynthe- through the additional role of COPI as a pro- tic cargo appears more concentrated. This is tective coat on cisternal membranes, i.e., BFA- achieved, at least in part, through a continuous induced dissociation of COPI coat from Golgi removal of ER export machinery components membranes enables uncontrolled formation of (e.g., p58, p24, and SNARE proteins) that are Golgi-derived tubules that within minutes fuse then returned to the ER via vesicular transport with the ER, collapsing most of the Golgi into intermediates controlled by the COPI coat the ER. Strikingly, removal of BFA equally rap- machinery. This apparent “distillation” process idly leads to the reformation of individual func- continues throughout the secretory pathway tional Golgi stacks positioned at the various from as far as the trans-part of the Golgi appa- ER exit sites and after microtubule-dependent ratus (Miesenbock and Rothman 1995). Wheth- transport, coalesce into the central and juxtanu- er or not such distillation occurs through intra- clear Golgi apparatus. Thus, the two organelles Golgi cisternal transport or through direct de- are temporally as well as spatially linked to en- livery to the ER is part of ongoing investigation, able productive communication and trafficking including the extent of recycling (see below). for the process of protein secretion, as well as The Golgi apparatus is morphologically dis- lipid biosynthesis. This link gives rise to some tinct from the ER because of its juxtanuclear unexpected functions (see below). position, and at the ultrastructural level, ap- pears as a ribbon-like structure of laterally in- ORGANELLE ISOLATION AND MASS terconnected stacks of flattened cisternae, each SPECTROMETRY having a network of extensive fenestrated mem- branes associated both at their cis- and trans- Isolation of the ER and Golgi apparatus from face. Biosynthetic cargo here, undergo extensive cultured cells, as well as the parenchyma of or- posttranslational modifications including the gans, has beenwell documented (Schneideret al. maturation of N-linked oligosaccharides, and 1953; Ernster et al. 1962; De Duve and Beaufay the addition of O-linked oligosaccharides. At 1981; Balch et al. 1984; Gilchrist et al. 2006). This the trans-face, cargo is sorted and packaged led to the uncovering of the molecular machin- for transport either to the plasma membrane ery for protein translocation across membranes 2 Cite this article as Cold Spring Harb Perspect Biol 2013;5:a015073 Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press ER–Golgi Proteomics (Blobel and Dobberstein 1975), the machines PEPTIDE MATCHING for protein folding and processing (De Lorenzo et al. 1966; Haas and Wabl 1983), and the ma- As shown with the distribution of 20 equimolar, chines for the egress of newly synthesized pro- highly purified human test proteins sent to 27 tein out of the ER for further processing and different international labs, regardless of the sorting in the Golgi apparatus (Zanetti et al. mass spectrometer and software used to match 2012). In this way, our knowledge base of the tandem mass spectra from fragmented tryp- translocon, cotranslational transport of proteins tic peptides to cognate peptide sequences, the across the ER membrane, the role of molecular match is usually (but not always) failsafe; how- chaperones, and the various coats and GTPases ever, subsequent matching to cognate proteins regulating the structure and functions of the ER remains difficult. Indeed, of the 27 labs, only and Golgi apparatus has been built. seven were able to do this correctly (Bell et al. For organelles, as for any other characteriza- 2009). This was not because the quality of the tion methodology (EM, marker enzyme, west- tandem mass spectra generated was poor. Rath- ern blot enrichment), organelle enrichment and er, inadequacies in databases and software for assessment of degree of homogeneity is a crite- matching peptides to proteins were mainly at rion well accepted by the subcellular fraction- fault. Even today, databases remain a major hur- ation community. For the ER and Golgi appara- dle for large-scale projects that depend on the tus, electron microscopy (especially of samples characterization of proteins by mass spectrom- processed by the Baudhuin filtration apparatus etry. When organelles are added to the mix, the [Baudhuin