Catherine E. Au

Quantitative Proteomics Analysis of the Secretory Pathway

We report more than 1400 proteins of the secretory-pathway proteome and provide spatial information on the relative presence of each protein in the rough and smooth ER Golgi cisternae and Golgi-derived COPI vesicles. The data support a role for COPI vesicles in recycling and cisternal maturation, showing that Golgi-resident proteins are present at a higher concentration than secretory cargo. Of the 1400 proteins, 345 were identified as previously uncharacterized. Of these, 230 had their subcellular location deduced by proteomics. This study provides a comprehensive catalog of the ER and Golgi proteomes with insight into their identity and function.

Sorting out glycosylation enzymes in the Golgi apparatus

The study of glycosylation and glycosylation enzymes has been instrumental for the advancement of Cell Biology. After Neutra and Leblond showed that the Golgi apparatus is the main site of glycosylation, elucidation of oligosaccharide structures by Baenziger and Kornfeld and subsequent mapping of glycosylation enzymes followed. This enabled development of an in vitro transport assay by Rothman and co‐workers using glycosylation to monitor intra Golgi transport which, complemented by yeast genetics by Schekman and co‐workers, provided much of the fundamental insights and key components of the secretory pathway that we today take for granted. Glycobiology continues to play a key role in Cell Biology and here, we look at the use of glycosylation enzymes to elucidate intra Golgi transport.

Cell biology through proteomics – ad astra per alia porci

Isolated subcellular fractions have been instrumental in elucidating cell function. The use of such fractions for the identification and biochemical characterization of subcellular organelles, combined with cell- free systems, has provided key insights into the function and machineries of organelles, including those involved in vesicle transport, quality control and protein sorting. Despite their obvious utility, popular cell biology has come to regard in vitro-based approaches as inferior to in vivo-based approaches. Usual criticisms are contamination, non-representative processes and an inability to recreate the dynamic processes seen in vivo. In a similar way, proteomics has been viewed with reservation. Despite this, and building on the tradition of in vitro-based approaches, organelle proteomics based on liquid chromatography and tandem mass-spectrometry has recently made significant contributions to cell biology, and now allows the molecular machineries of organelles to be defined with high precision.

Expression, sorting, and segregation of Golgi proteins during germ cell differentiation in the testis

A total of 1318 proteins characterized in the male germ cell Golgi apparatus reveal a new germ cell–specific Golgi marker and a new pan-Golgi marker for all cells. The localization of these and other Golgi proteins reveals differential expression linked to mitosis, meiosis, acrosome formation, and postacrosome Golgi migration and destination in the late spermatid.

Compartmentalization of membrane trafficking, glucose transport, glycolysis, actin, tubulin and the proteasome in the cytoplasmic droplet/Hermes body of epididymal sperm.

Discovered in 1909 by Retzius and described mainly by morphology, the cytoplasmic droplet of sperm (renamed here the Hermes body) is conserved among all mammalian species but largely undefined at the molecular level. Tandem mass spectrometry of the isolated Hermes body from rat epididymal sperm characterized 1511 proteins, 43 of which were localized to the structure in situ by light microscopy and two by quantitative electron microscopy localization. Glucose transporter 3 (GLUT-3) glycolytic enzymes, selected membrane traffic and cytoskeletal proteins were highly abundant and concentrated in the Hermes body. By electron microscope gold antibody labelling, the Golgi trafficking protein TMED7/p27 localized to unstacked flattened cisternae of the Hermes body, as did GLUT-3, the most abundant protein. Its biogenesis was deduced through the mapping of protein expression for all 43 proteins during male germ cell differentiation in the testis. It is at the terminal step 19 of spermiogenesis that the 43 characteristic proteins accumulated in the nascent Hermes body.

Cell Biology of the Endoplasmic Reticulum and the Golgi Apparatus through Proteomics

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.