Faculty Bibliography

Rab GTPases regulate membrane trafficking at the stages of vesicle formation, movement and fusion with target compartments. Rab11 GTPase coordinates trafficking at biosynthetic and endocytic recycling routes acting at the trans-Golgi network (TGN), post-Golgi vesicles and recycling endosomes. Rab11 Binding Protein (Rab11BP) has long been described as a potential Rab11 effector but its function remains unknown. The structure of Rab11BP includes a Rab11 binding domain and several domains presumably involved in protein-protein interactions, which include an FFAT-like domain, a proline rich domain and seven WD40 repeats typical of scaffold proteins. Here we used shRNA silencing experiments to first evaluate whether Rab11BP is involved in the Rab11-dependent endocytic recycling of transferring receptor (TfR) and then assessed the protein traffic between the endoplasmic reticulum (ER) and Golgi. We silenced Rab11BP expression with shRNA using lentiviral transduction or microinjection. Rab11BP-silenced cells showed normal TfR endocytosis and recycling analyzed by FACS. However, the distribution of KDELR-GFP and the retrograde-impaired mutant KDELR(D193N)-GFP indicated that Rab11BP functions in Golgi-to-ER retrograde trafficking. Rab11BP silencing led the KDELRGFP to change its distribution from a predominant ER location to an accumulation at the cis-Golgi, colocalizing with Giantin, while the Golgi-retained mutant KDELR(D193N)-GFP remained unaffected. This indicates an impaired retrograde Golgi-to-ER transport without affecting the anterograde transport from ER to Golgi, which likely impact on the ER function of KDEL-bearing chaperones. Rab11BP silencing decreased TGN46, furin, M6PR and calnexin protein levels and induced the characteristic fragmentation of the TGN associated with an impaired Golgi-to-ER transport. These results indicate that Rab11BP is required for retrograde transport from the Golgi-to-ER contributing to the maintenance of the Golgi structure and homeostasis. As to our knowledge Rab11 has not been involved in this step of the biosynthetic trafficking, our results suggest that Rab11BP might have functions independently of Rab11

Uroplakins (UPs) are major differentiation products of urothelial umbrella cells, playing important roles in forming the permeability barrier, and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic E. coli receptor. While it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immuno-microscopy of normal and mutant mouse urothelia showed that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently, a Rab27b/Slp2-a complex mediated FV-membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also showed that keratin 20 (K20), which formed a chicken-wire network 150-300 nm below the apical membrane and had hole sizes allowing FV passage, defined a subapical compartment containing FVs primed and strategically located for fusion. Finally, we showed that Rab8/11 and Rab27b function in the same pathway, that Rab27b-knockout leads to uroplakin and Slp2-a destabilization, and that Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking.

Despite a high degree of structural homology and shared exchange factors, effectors and GTPase activating proteins, a large body of evidence suggests functional heterogeneity among Ras isoforms. One aspect of Ras biology that may explain this heterogeneity is the differential subcellular localizations driven by the C-terminal hypervariable regions of Ras proteins. Spatial heterogeneity has been documented at the level of organelles: palmitoylated Ras isoforms (H-Ras and N-Ras) localize on the Golgi apparatus whereas K-Ras4B does not. We tested the hypothesis that spatial heterogeneity also exists at the sub-organelle level by studying the localization of differentially palmitoylated Ras isoforms within the Golgi apparatus. Using confocal, live-cell fluorescent imaging and immunogold electron microscopy we found that, whereas the doubly palmitoylated H-Ras is distributed throughout the Golgi stacks, the singly palmitoylated N-Ras is polarized with a relative paucity of expression on the trans Golgi. Using palmitoylation mutants, we show that the different sub-Golgi distributions of the Ras proteins are a consequence of their differential degree of palmitoylation. Thus, the acylation state of Ras proteins controls not only their distribution between the Golgi apparatus and the plasma membrane, but also their distribution within the Golgi stacks. J. Cell. Physiol. 230: 610-619, 2015. (c) 2014 Wiley Periodicals, Inc., A Wiley Company.

I entered the field of cell biology during its formative phase and contributed technical advances in electron microscopy that gave new insights into the physiological roles of subcellular compartments. I concentrated for many years on the structure and function of the rough endoplasmic reticulum where I aimed at explaining mechanistically how the functional specialization of ribosomes bound to the endoplasmic reticulum membranes is achieved. This work eventually led to the formulation of the signal hypothesis with Gunter Blobel in 1971. Subsequently, my laboratory contributed to the birth of the field of protein traffic with the demonstration that membrane-bound ribosomes in the ER are also responsible for the synthesis of membrane and luminal proteins destined to other subcellular compartments, pointing to the existence of sorting signals in the newly synthesized polypeptides and corresponding discriminating trafficking mechanisms. I have derived great satisfaction from the fact that some of the work I will present, including the introduction with M. Cerejido of the MDCK cell line to study the development and properties of polarized epithelia and the discovery with Rodriguez-Boulan of the polarized budding of enveloped viruses, has helped many laboratories to continue to explore the fascinating mechanisms that contribute to generate the complex organization of eukaryotic cells

There are various procedures for isolating microsomal fractions from tissue culture cells. The essential conditions for each step of one procedure are described here. Notes for special circumstances are included so that the procedure can be modified according to the experimental purpose.

When eukaryotic cells are homogenized, the rough endoplasmic reticula are converted into small vesicles, called rough microsomes. Strategies for the isolation of rough microsomes are introduced here, as are methods for evaluating the purity and intactness of an isolated rough microsomal fraction.

This protocol describes how to prepare rat liver rough microsomes that contain undegraded membrane-bound polysomes and can function very well in an in vitro translation system. It uses endogenous ribonuclease inhibitor in all steps, avoiding pelleting rough microsomes in all steps and sacrificing good recovery.

This protocol describes how to prepare rough microsomes from dog pancreas. These microsomes can be used to analyze mechanisms of protein translocation and membrane insertion.

Mitochondrial respiratory chain disorders are characterized by loss of electron transport chain (ETC) activity. Although the causes of many such diseases are known, there is a lack of effective therapies. To identify genes that confer resistance to severe ETC dysfunction when inactivated, we performed a genome-wide genetic screen in haploid human cells with the mitochondrial complex III inhibitor antimycin. This screen revealed that loss of ATPIF1 strongly protects against antimycin-induced ETC dysfunction and cell death by allowing for the maintenance of mitochondrial membrane potential. ATPIF1 loss protects against other forms of ETC dysfunction and is even essential for the viability of human rho degrees cells lacking mitochondrial DNA, a system commonly used for studying ETC dysfunction. Importantly, inhibition of ATPIF1 ameliorates complex III blockade in primary hepatocytes, a cell type afflicted in severe mitochondrial disease. Altogether, these results suggest that inhibition of ATPIF1 can ameliorate severe ETC dysfunction in mitochondrial pathology.