ErbB2 is a transmembrane tyrosine kinase whose surface overexpression is linked to tumorigenesis and poor prognosis in breast cancer patients. Two models have emerged that account for the high surface distribution of ErbB2. In one model, the surface pool is dynamic and governed by a balance between endocytosis and recycling, whereas in the other it is retained, static, and excluded from endocytosis. These models have contrasting implications for how ErbB2 exerts its biological function and how cancer therapies might down-regulate surface ErbB2, such as the antibody trastuzumab (Herceptin) or the Hsp90 inhibitor geldanamycin. Little is known, however, about how these treatments affect ErbB2 endocytic trafficking. To investigate this issue, we examined breast carcinoma cells by immunofluorescence and quantitative immunoelectron microscopy and developed imaging and trafficking kinetics assays using cell surface fluorescence quenching. Surprisingly, trastuzumab does not influence ErbB2 distribution but instead recycles passively with internalized ErbB2. By contrast, geldanamycin down-regulates surface ErbB2 through improved degradative sorting in endosomes exclusively rather than through increased endocytosis. These results reveal substantial dynamism in the surface ErbB2 pool and clearly demonstrate the significance of endosomal sorting in the maintenance of ErbB2 surface distribution, a critical feature of its biological function.

Rab GTPases are localized to various intracellular compartments and are known to play important regulatory roles in membrane trafficking. Here, we report the subcellular distribution and function of Rab14. By immunofluorescence and immunoelectron microscopy, both endogenous as well as overexpressed Rab14 were localized to biosynthetic (rough endoplasmic reticulum, Golgi, and trans-Golgi network) and endosomal compartments (early endosomal vacuoles and associated vesicles). Notably overexpression of Rab14Q70L shifted the distribution toward the early endosome associated vesicles, whereas the S25N and N124I mutants induced a shift toward the Golgi region. A similar, although less pronounced, redistribution of the transferrin receptor was also observed in cells overexpressing Rab14 mutants. Impairment of Rab14 function did not however affect transferrin uptake or recycling kinetics. Together, these findings suggest that Rab14 is involved in the biosynthetic/recycling pathway between the Golgi and endosomal compartments.

Endocytosis is crucial for various aspects of cell homeostasis. Here, we show that proapoptotic death receptors (DRs) trigger selective destruction of the clathrin-dependent endocytosis machinery. DR stimulation induced rapid, caspase-mediated cleavage of key clathrin-pathway components, halting cellular uptake of the classic cargo protein transferrin. DR-proximal initiator caspases cleaved the clathrin adaptor subunit AP2α between functionally distinct domains, whereas effector caspases processed clathrin’s heavy chain. DR5 underwent ligand-induced, clathrin-mediated endocytosis, suggesting that internalization of DR signaling complexes facilitates clathrin-pathway targeting by caspases. An endocytosis-blocking, temperature-sensitive dynamin-1 mutant attenuated DR internalization, enhanced caspase stimulation downstream of DRs, and increased apoptosis. Thus, DR-triggered caspase activity disrupts clathrin-dependent endocytosis, leading to amplification of programmed cell death.

To delineate the role of the melanocyte lineage-specific protein Melan-A/MART-1 in melanogenic functions, a set of biochemical and microscopical studies was performed. Biochemical analysis revealed that Melan-A/MART-1 is post-translationally acylated and undergoes a rapid turnover in a pigmented melanoma cell line. Immunofluorescence and immunoelectron microscopy analyses indicated that Melan-A/MART-1 is mainly located in the Golgi area and only partially colocalizes with melanosomal proteins. Quantitative immunoelectron microscopy showed that the highest proportion of the cellular content of Melan-A/MART-1 was found in small vesicles and tubules throughout the cell, whereas the concentration was maximal in the Golgi region, particularly the trans-Golgi network. Substantial labeling was also present on melanosomes, endosomes, ER, nuclear envelope, and plasma membrane. In early endosomes, Melan-A was enriched in areas of the limiting membrane covered by a bi-layered coat, a structural characteristic of melanosomal precursor compartments. Upon melanosome maturation, Melan-A concentration decreased and its predominant localization shifted from the limiting membrane to internal vesicle membranes. In conjunction with its acylation, the high expression levels of Melan-A in the trans-Golgi network, in dispersed vesicles, and on the limiting membrane of premelanosomes indicate that the protein may play a role during the early stage of melanosome biogenesis. Melan-A/MART-1 (hereafter designated as Melan-A) was originally identified by functional screening of expression libraries using melanoma-reactive cytolytic T lymphocytes (CTL) isolated from melanoma patients (1,2). Its expression is restricted to melanin-producing cells, including normal and transformed skin melanocytes and retinal pigment epithelium (2). Approximately 90% of primary and 80% of metastatic melanoma tumors express Melan-A transcripts. The homologous mouse protein shares 69% identity with the human one (3). The human cDNA codes for an intracellular protein of 118 amino acids with an apparent molecular weight of 22-24 kDa. An immunodominant CTL epitope corresponding to the amino acid sequence 26-35/27-35 and binding to HLA-A2 molecules has been identified (4,5).Because of the wide distribution of Melan-A in melanoma tumors and the fact that specific CTL responses can be detected frequently in melanoma patients, this protein has received much attention in the field of tumor immunology and it is the target of many vaccination trials (6,7). In spite of the wealth of information on the immunogenicity of Melan-A, little is known on the biological properties of this protein. The highly restricted expression pattern suggests a specific role in pigmented cells. The protein contains a transmembrane domain spanning amino acids 27-47 and is devoid of consensus signals for N-linked glycosylation. We have recently shown that Melan-A is inserted in membranes with the amino terminus facing the lumen of the exocytic compartment and the longer carboxy terminus exposed t...

BackgroundAPOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs.MethodsImmunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies.ResultsBoth endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0, -G1, and -G2 showed no differences in localization among variants.ConclusionsAPOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues.